Multiparametric fluorescence in situ hybridization

ABSTRACT

The invention relates to a set of combinatorially labeled oligonucleotide probes each member thereof: (i) having a predetermined label distinguishable from the label of any other member of said set, and (ii) being capable of specifically hybridizing with a predetermined chromosome or nucleic acid molecule, and to the use of such molecules, alone or in concert with nucleic acid amplification methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 09/468,823, filed Dec. 22, 1999, a continuation-in-part of application Ser. No. 09/088,845, filed Jun. 2, 1998, a continuation-in-part of application Ser. No. 09/088,087, filed Jun. 1, 1998, now U.S. Pat. No. 6,007,994 a continuation-in-part of application Ser. No. 08/640,657, filed May 1, 1998 now U.S. Pat. No. 5,759,781, a continuation-in-part of application Ser. No. 08/580,717, filed Dec. 29, 1995, now abandoned and a continuation-in-part of application Ser. No. 08/577,622, filed Dec. 22, 1995 now abandened.

FIELD OF THE INVENTION

The present invention relates to nucleic acid chemistry, and more specifically to reagents and methods for accomplishing multiplex image analysis of chromosomes and chromosomal fragments. The invention may be used to diagnose chromosomal abnormalities, infectious agents, etc. This invention was made in part using Government funds. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

The determination of the presence and condition of chromosomes and chromosomal fragments in a biological sample is of immense importance in the diagnosis of disease. Traditionally, such determinations have been done manually by inspecting metaphase chromosomal preparations that have been treated with specialized stains to reveal characteristic banding patterns. Unfortunately, the interpretation of such banding patterns requires substantial skill and is technically difficult. Hence, alternate methods of analyzing chromosomal presence and arrangement have been sought.

One alternative approach to the problem of chromosome identification has involved the use of labeled chromosome-specific oligonucleotide probes to label repetitive sequences of interphase chromosomes (Cremer, T. et al., Hum. Genet. 74:346-352 (1986); Cremer, T. et al., Exper. Cell Res. 176:119-220 (1988)). Such methods have been shown to be useful in the prenatal diagnosis of Down's Syndrome, as well as in the detection of chromosomal abnormalities associated with tumor cell lines. Chromosome-specific probes of repetitive DNA that localize to discrete sub-regions of a chromosome are, however, unsuitable for analyses of many types of chromosomal abnormalities (e.g., translocations or deletions).

Ward, D. C. et al. (PCT Application WO/05789, herein incorporated by reference) discloses a chromosomal in situ suppression (“CISS”) hybridization method for specifically labeling selected mammalian chromosomes in a manner that permits the recognition of chromosomal aberrations. In that method, sample DNA is denatured and permitted to hybridize with a mixture of fluorescently labeled chromosome-specific probes having high genetic complexity and unlabeled non-specific competitor probes. Chromosomal images were obtained as described by Manuelidis, L. et al. (Chromosoma 96:397-410 (1988), herein incorporated by reference). The method provides a rapid and highly specific assessment of individual mammalian chromosomes. The method permits, by judicious selection of appropriate probes and/or labels, the visualization of sub-regions of some or all of the chromosomes in a preparation. For example, by using more than one probe, each specific for a sub-region of a target chromosome, the method permits the simultaneous analysis of several sub-regions on that chromosome. The number of available fluorophores limits the number of chromosomes or chromosomal sub-regions that can be simultaneously visualized.

As described in PCT Application WO/05789, a “combinatorial” variation of the CISS method can be employed. In the simplest case, two fluors permit three different chromosomes or chromosomal sub-regions to be simultaneously visualized. In this variation, a hybridization probe mixture is made from a single set of probe sequences composed of two halves, each separately labeled with a different fluorophore. Upon hybridization, the two fluorophores produce a third fluorescence signal that is optically distinguishable from the color of the individual fluorophores. Extension of this approach to Boolean combinations of n fluorophores permits the labeling of 2^(n)−1 chromosomes.

Ried, T. et al. (Proc. Natl. Acad. Sci. (U.S.A.) 89:1388-1392 (1992), herein incorporated by reference) describes the use of an epi-fluorescent microscope equipped with a digital imaging camera and computer software to “pseudocolor” the fluorescence patterns obtained from simultaneous in situ hybridization with seven probes using three fluorophores. The use of wavelength-selective filters allows one to isolate and collect separate gray scale images of each fluorophore. These images can be subsequently merged via appropriate software. The sensitivity and linearity of CCD cameras surmounts the technical difficulties inherent in color film-based photomicroscopy.

Although such efforts have increased the number of chromosomes that can be simultaneously detected and analyzed using in situ hybridization methods, it would be highly desirable to define a set of fluorophores having distinguishable emission spectra to permit the simultaneous detection and analysis of large numbers of different chromosomes and chromosomal sub-regions. The present invention provides such reagents as well as methods and apparatus for their use.

SUMMARY OF THE INVENTION

The invention concerns reagents and methods for combinatorial labeling of nucleic acid probes sufficient to permit the visualization and simultaneous identification of all 22 autosomal human chromosomes and the human X and Y chromosomes, or defined sub-regions thereof. Such specific labeling of entire chromosomes or defined sub-regions thereof is referred to as “painting.” The invention further concerns reagents and methods for combinatorial labeling of nucleic acid probes sufficient to permit the characterization of bacteria, viruses and/or lower eukaryotes that may be present in a clinical or non-clinical preparation.

In detail, the invention concerns a set of combinatorially labeled oligonucleotide probes comprised of a first and a second subset of probes, wherein:

(A) each member of the first subset of probes comprises a plurality of an oligonucleotide: (i) being linked or coupled to a predetermined label distinguishable from the label of any other member of the first or second subsets of probes, and (ii) being capable of specifically hybridizing with one predetermined autosomal or sex chromosome of a human karyotype;

the first subset of probes set having sufficient members to be capable of specifically hybridizing each autosomal or sex chromosome of the human karyotype to at least one member; and

(B) each member of the second subset of probes comprises a plurality of an oligonucleotide: (i) being linked or coupled to a predetermined label distinguishable from the label of any other member of the first or second subset, and (ii) being capable of specifically hybridizing with one extra-chromosomal polynucleotide copy of a predetermined region of an autosomal or sex chromosome of the human karyotype.

The invention further concerns a set of combinatorially labeled oligonucleotide probes comprised of a first subset of genotypic probes and a second subset of phenotypic probes, wherein:

(A) each member of the first subset of genotypic probes comprises a plurality of an oligonucleotide: (i) being linked or coupled to a predetermined label distinguishable from the label of any other member of the first or second subsets of probes, and (ii) being capable of specifically hybridizing with a region of a nucleic acid of a preselected bacterium, virus or lower eukaryote;

the first subset of probes set having sufficient members to be capable of distinguishing the preselected bacterium, virus, or lower eukaryote from other bacteria, viruses, or lower eukaryotes; and

(B) each member of the second subset of phenotypic probes comprises a plurality of an oligonucleotide: (i) being linked or coupled to a predetermined label distinguishable from the label of any other member of the first or second subset, and (ii) being capable of specifically hybridizing with a predetermined polynucleotide region of the chromosome of the preselected bacterium, virus, or lower eukaryote, or an extra-chromosomal copy thereof so as to permit the determination of whether the preselected bacterium, virus, or lower eukaryote exhibits a preselected phenotype.

The invention additionally concerns a method of simultaneously identifying and distinguishing the individual autosomal and sex chromosomes of a human karyotype which comprises the steps:

(I) contacting a preparation of the chromosomes, in single-stranded form, under conditions sufficient to permit nucleic acid hybridization to occur with a set of combinatorially labeled oligonucleotide probes comprised of a first and a second subset of probes, wherein:

(A) each member of the first subset of probes comprises a plurality of an oligonucleotide: (i) being linked or coupled to a predetermined label distinguishable from the label of any other member of the first or second subsets of probes, and (ii) being capable of specifically hybridizing with one predetermined autosomal or sex chromosome of a human karyotype;

the first subset of probes set having sufficient members to be capable of specifically hybridizing each autosomal or sex chromosome of the human karyotype to at least one member; and

(B) each member of the second subset of probes comprises a plurality of an oligonucleotide: (i) being linked or coupled to a predetermined label distinguishable from the label of any other member of the first or second subset, and (ii) being capable of specifically hybridizing with an a predetermined extra-chromosomal polynucleotide copy of a region of an autosomal or sex chromosome of the human karyotype.

(II) for each chromosome of the preparation hybridized to a member of the first subset of probes, detecting and identifying the predetermined label of that member and correlating the identity of the label of that member with the identity of the autosomal or sex chromosome of the human karyotype with which that member specifically hybridizes, to thereby identify the chromosome hybridized to the member;

(III) repeating step (II) until each autosomal and sex chromosome of the human karyotype has been identified in the preparation

(IV) for each member of the second subset of probes hybridized to a predetermined extra-chromosomal polynucleotide copy of a region of an autosomal or sex chromosome detecting and identifying the predetermined label of that member and correlating the identity of the label of that member with the identity of the region of the autosomal or sex chromosome of the human karyotype with which that member specifically hybridizes, to thereby identify the region of the autosomal or sex chromosome hybridized to the member;

(V) repeating step (IV) for each member of the second subset of probes.

The invention additionally concerns a method of simultaneously identifying and distinguishing a preselected bacterium, virus, or lower eukaryote from other bacteria, viruses or lower eukaryotes that may be present in a sample which comprises the steps:

(I) contacting a preparation suspected to contain the preselected bacterium, virus, or lower eukaryote, under conditions sufficient to permit in situ nucleic acid hybridization to occur, with a set of combinatorially labeled oligonucleotide probes comprised of a first subset of genotypic probes and a second subset of phenotypic probes, wherein:

(A) each member of the first subset of genotypic probes comprises a plurality of an oligonucleotide: (i) being linked or coupled to a predetermined label distinguishable from the label of any other member of the first or second subsets of probes, and (ii) being capable of specifically hybridizing with a region of a nucleic acid of the preselected bacterium, virus or lower eukaryote;

the first subset of probes set having sufficient members to be capable of distinguishing the preselected bacterium, virus, or lower eukaryote from other bacteria, viruses, or lower eukaryotes present in the preparation; and

(B) each member of the second subset of probes comprises a plurality of an oligonucleotide: (i) being linked or coupled to a predetermined label distinguishable from the label of any other member of the first or second subset, and (ii) being capable of specifically hybridizing with a predetermined polynucleotide region of the nucleic acid of the preselected bacterium, virus, or lower eukaryote, or an extra-chromosomal copy thereof so as to permit the determination of whether the preselected bacterium, virus, or lower eukaryote exhibits a preselected phenotype.

(II) for each member of the first subset of probes hybridized to a region of a chromosome of a preselected bacterium, virus or lower eukaryote, detecting and identifying the predetermined label of that member and correlating the identity of the label of that member with the identity of the bacterium, virus or lower eukaryote with which that member specifically hybridizes, to thereby identify the bacterium, virus or lower eukaryote hybridized to the member;

(III) repeating step (II) for each member of the first subset of probes;

(IV) for each member of the second subset of probes hybridized to a predetermined polynucleotide region of the chromosome of the preselected bacterium, virus, or lower eukaryote, or an extra-chromosomal copy thereof, detecting and identifying the predetermined label of that member and correlating the identity of the label of that member with the identity of the predetermined region, to thereby identify the presence of the predetermined region on a chromosome of the preselected bacteria, virus or lower eukaryote;

(V) repeating step (IV) for each member of the second subset of probes.

The invention particularly contemplates the embodiments in which the members of the above sets of probes are detectably labeled with fluorophores, and, wherein at least one member of the set is combinatorially labeled with either one, two, three, four or five fluorophores selected from the group consisting of the fluorophores FITC, Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, and wherein each member of the set is labeled with at least one fluorophore selected from the fluorophore group.

The invention additionally provides a set of combinatorially labeled oligonucleotide probes, each member thereof: (i) having a predetermined label distinguishable from the label of any other member of the set, and (ii) being capable of specifically hybridizing with a telomeric region of one predetermined autosomal or sex chromosome of a human karyotype; the set having sufficient members to be capable of specifically hybridizing each autosomal or sex chromosome of the human karyotype to at least one member.

The invention additionally provides a method of simultaneously identifying and distinguishing the individual autosomal and sex chromosomes of a human karyotype which comprises the steps:

(a) contacting a preparation of the chromosomes, in single-stranded form, under conditions sufficient to permit nucleic acid hybridization to occur with a set of combinatorially labeled oligonucleotide probes, each member thereof: (i) having a predetermined label distinguishable from the label of any other member of the set, and (ii) being capable of specifically hybridizing with a telomeric region of one predetermined autosomal or sex chromosome of a human karyotype; the set having sufficient members to be capable of specifically hybridizing each autosomal or sex chromosome of the human karyotype to at least one member; wherein the contacting thereby causes at least one of each autosomal or sex chromosome of the preparation to become hybridized to at least one member of the set of probes;

(b) for each chromosome of the preparation hybridized to a member of the set of probes, detecting and identifying the predetermined label of that member and correlating the identity of the label of that member with the identity of the autosomal or sex chromosome of the human karyotype with which that member specifically hybridizes, to thereby identify the chromosome hybridized to the member; and

(c) repeating step (b) until each autosomal and sex chromosome of the human karyotype has been identified in the preparation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic illustration of a CCD camera and microscope employed in accordance with the present methods.

FIG. 2 shows the raw data from a karyotypic analysis of chromosomes from a bone marrow patient (BM2486). Adjacent to each source image is a chromosome “mask” generated by the software program. In FIG. 2, panels A and B are the DAPI image and mask; panels C and D are FITC image and mask; panels E and F are Cy3 image and mask; panels G and H are Cy3.5 image and mask; panels I and J are Cy5 image and mask; and panels K and L are Cy7 image and mask.

FIGS. 3A and 3B show the identification of individual chromosomes by spectral signature of patient BM2486. FIG. 2 is the same photograph as FIG. 3A, except that it is gray scale pseudocolored. FIG. 3B displays the karyotypic array of the chromosomes.

FIG. 4 shows the differentiation of bacteria by in situ hybridization. Panels A-E represent in situ hybridization assay results on a laboratory derived mixture of three bacteria (F. nucleatum, A. actinomycetemcomitans and E. corrodens) using a mixture of genomic DNA probes for each organism. Panel A shows all the bacteria present in the microscope field detected by (DAPI), a general DNA binding fluorophore. Panel B shows F. nucleatum using Cascade Blue detection. Panel C shows A. actinomycetemcomitans using FITC detection. Panel D shows E. corrodens using Rhodamine detection. Panel E is a computer-merged composite of panels B-D showing the differentiation of each bacteria in the sample. Panel F is a similar analysis of a mixture of C. gingivalis (using rhodamine detection) and P. intermedia (using FITC detection) when hybridized with a combination of genomic DNA probes for those organisms. Panel G shows an in situ hybridization assay for a combination of seven different bacteria hybridized with a mixture of seven genomic DNA probes. In panel G, the numbers (1)-(7) identify the bacteria. F. nucleatum (1), E. corrodens (2) and A. actinomycetemcomitans (3) are shown in blue (Cascade Blue detection). P. gingivalis (4) and C. ochracea (5) are shown in red (Rhodamine detection), whereas P. intermedia (6) and C. gingivalis (7) are seen in yellow (FITC detection). Panel H represents an in situ hybridization assay for the presence of A. actinomycetemcomitans (using FITC detection) in a plaque sample obtained from a patient with localized juvenile periodontist.

FIG. 5A shows a normal male metaphase chromosomal spread after hybridization with a 24-color set of telomere-specific probes, shown as a pseudocolorized image. FIG. 5B shows the final karyotype generated on the basis of the boolean spectral signature of the telomere-specific probes.

FIG. 6A shows the hybridization pattern of the chromosome 8 subtelomeric YACs (telomere-specific probes) on a normal metaphase chromosomal pread. FIG. 6B shows the hybridization pattern of the same probes (as those used in FIG. 6A) on a metaphase chromosomal spread from a patient with a myeloproliferative disorder. Previous cytogenetic analysis of this patient using G-banding revealed a trisomy 8 as the only change; the M-FISH telomere-specific probes show a split telomere signal on the short arms of two of the three chromosome 8, indicating an additional change: an inversion in this region.

DESCRIPTION OF THE PREFERRED EMBODIMENTS A. Overview of the Invention

Fluorescence in situ hybridization (FISH) is used in a variety of areas of research and clinical diagnostics (Gray, J. W. et al., Curr Opin Biotech 3:623-631 (1992); Xing, Y. et al., In: The Causes and Consequences of Chromosomal Aberrations. I.R. Kirsch Ed. CRC Press, Boca Raton, pages 3-28 (1993)). For the study of the chromosomal and suprachromosomal organization of the cell nucleus it is an indispensable tool (Cremer, T. et al., In: Cold Spring Harbor Symposia on Quantitative Biology, Volume LVIII, pp. 777-792, Cold Spring Harbor Laboratory Press, NY (1994)). Most importantly FISH offers the capacity for multiparameter discrimination. This allows the simultaneous visualization of several DNA probes using either a combinatorial (Nederlof, P. M. et al., Cytometry 10:20-27 (1989); Nederlof, P. M. et al., Cytometry 11:126-131 (1990); Ried, T. et al., Proc Natl Acad Sci (U.S.A.) 89:1388-1392 (1992a); Ried, T. et al., Hum Mol Genet 1:307-313 (1992b); Lengauer, C. et al., Hum Mol Genet 2:505-512 (1993); Popp, S. et al., Human Genetics 92:527-532 (1993); Wiegant, J. et al., Cytogenet Cell Genzet 63:73-76 (1993)) or a ratio labeling (Dauwerse, J. G. et al., Hum Mol Genet 1:593-598(1992), Nederlof, P. M. et al., Cytometry 13:839-845 (1992); du Manoir, S. et al., Hum Genet 90:590-610 (1993)) strategy. Up to twelve DNA probes have been visualized (Dauwerse, J. G. et al., Hum Mol Genet 1:593-598 (1992)). Consequently, the goal of 24 different colors has long been sought (Ledbetter, D. H., Hum Mol Genet 5:297-299 (1992)). Twenty-four different colors are an important threshold because they would allow the simultaneous visualization of the 22 autosomes and both sex chromosomes. Beside improved karyotyping, the possibility of simultaneously hybridizing 24 different and distinguishable DNA probes would allow the addressing of a large number of important biological questions. However, the previously published multicolor systems lacked the versatility for an extension to 24 colors and only proof-of-principle experiments were ever published.

The present invention results, in part, from the realization of multiparametric fluorescence in situ hybridization to achieve the simultaneous visualization of 24 different genetic targets with a combinatorial labeling strategy. This strategy permits discrimination between many more target sequences than there are spectrally distinguishable labels. The simplest way to implement such labeling is using a simple “Boolean” combination, i.e., a fluor is either completely absent (i.e. the value of “0” will be assigned) or present in unit amount (value of 1). For a single fluor A, there is only one useful combination (A=1) and for two fluors A and B, there are 3 useful combinations (A=1/B=0; A=0/B=1; A=1/B=1). There are 7 combinations of 3 fluors, 15 combinations of 4 fluors, 31 combinations of 5 fluors, 63 combinations of 6 fluors, and so on (n fluorophores permitting the labeling of 2^(n)−1 chromosomes). Thus, to uniquely identify all 24 chromosome types in the human genome using chromosome painting probe sets, only 5 distinguishable fluors are needed (31 total combinations). If each probe set is labeled with one or more of five spectrally distinct fluorophores in a combinatorial fashion, simple Boolean combination can be used to identify each DNA probe by a spectral signature dictated by its fluorophore composition (Speicher, M. R. et al, Nature Genet. 12:368-375 (1996), which reference is herein incorporated by reference).

B. Terminology of the Invention

The invention concerns a set of combinatorially labeled oligonucleotide probes, each member thereof: (i) having a predetermined label distinguishable from the label of any other member of the set, and (ii) being capable of specifically hybridizing with one predetermined autosomal or sex chromosome of a human karyotype. In the most preferred embodiment, the set will have a sufficient number of members to be capable of specifically and distinguishably hybridizing each autosomal or sex chromosome of said human karyotype to at least one member. As used herein, the term “karyotype” denotes the compliment of chromosomes found in a normal or aberrant cell. In a normal cells, the number of chromosomes is 46, comprising 22 pairs of autosomal chromosomes and 2 sex chromosomes (either 2 X chromosomes (if female) or an X and Y chromosome (if male)). The labels are said to be distinguishable in that the particular label of any one member of the set (and the identity of that member) differ from the particular label and identity of any other member of the set. Since each probe member is capable of specifically hybridizing to only one chromosome (or sub-chromosomal region) and since the identity of the label and probe are known in advance, the detection of a particular label associated with an unidentified chromosomal region means that the probe bearing that label has become hybridized to the unidentified chromosomal region. Since the chromosome to which that probe specifically hybridizes is known, the detection of a distinguishable label permits the identification of the chromosomal region.

More specifically, the invention concerns fluors that can be used to label oligonucleotide probes so that such probes may be used in multiparametric fluorescence in situ hybridization. As used herein, a “fluor” or “fluorophore” is a reagent capable of emitting a detectable fluorescent signal upon excitation. Most preferably, the fluor is coupled directly to the pyrimidine or purine ring of the nucleotides of the probe (Ried, T. et al. (Proc. Natl. Acad. Sci. (U.S.A.) 89:1388-1392 (1992), herein incorporated by reference; U.S. Pat. Nos. 4,687,732; 4,711,955; 5,328,824; and 5,449,767, each herein incorporated by reference. Alternatively, the fluor may be indirectly coupled to the nucleotide, as for example, by conjugating the fluor to a ligand capable of binding to a modified nucleotide residue. The most preferred ligands for this purpose are avidin, streptavidin, biotin-binding antibodies and digoxigenin-binding antibodies. Methods for performing such conjugation are described by Pinkel, D. et al., Proc. Nat'l. Acad. Sci. (U.S.A.) 83:2934-2938 (1986), herein incorporated by reference).

The term “multiparametric fluorescence” denotes the combinatorial use of multiple fluors to simultaneously label the same chromosome or sub-chromosomal fragment, and their detection and characterization. Chromosomes or sub-chromosomal fragments are said to be simultaneously labeled if they are exposed to more than a single chromosome-specific probe under conditions sufficient to permit each chromosome-specific probe to independently hybridize to its target chromosome. As used herein, it is thus unnecessary for all such hybridization reactions to commence and conclude at the same instant. The simultaneous labeling permitted by the present invention is thus in contrast to protocols in which chromosomes are exposed to only a single chromosome-specific probe at a time.

The simultaneous detection and characterization permitted by the present invention denotes an ability to detect multiple (and most preferably all) of the autosomal and/or sex chromosomes in a sample, without any need to add further reagent, or probe after the detection of the first chromosome.

In the simplest embodiment, digital images of the chromosomes are obtained for each fluorophore employed, thereby providing a series of gray scale fluorescence intensities associated with each fluorophore and each chromosome. The final image is obtained by pseudocoloring the blended gray scale intensities for each chromosome.

The invention thus provides a method of simultaneously identifying and distinguishing the individual autosomal and sex chromosomes of a human karyotype which comprises contacting a preparation of chromosomes, that has been previously treated to render it in single-stranded form, with the above-described set of combinatorially labeled oligonucleotide probes, under conditions sufficient to permit nucleic acid hybridization to occur.

Such treatment causes at least one of each autosomal or sex chromosome of the preparation to become hybridized to at least one member of said set of probes. For each chromosome of the preparation hybridized to a member of the set of probes one next detects and identifies the predetermined label of that member and correlates the identity of the label of that member with the identity of the autosomal or sex chromosome of said human karyotype with which that member specifically hybridizes. This process identifies the chromosome hybridized to the member. This last step is repeated until each or a desired number of autosomal and sex chromosome of the human karyotype has been identified in the preparation.

The oligonucleotide probes used in accordance with the methods of the present invention are of either of two general characteristics. In one embodiment, such probes are chromosome or sub-chromosome specific (i.e., they hybridize to DNA of a particular chromosome at lower c_(o)t^(1/2) than with DNA of other chromosomes; c_(o)t^(1/2) being the time required for one half of an initial concentration (c_(o)) of probe to hybridize to its complement). Alternatively, such probes are feature (e.g., telomere, centromere, etc.) specific. Such probes, being proximal to the telomere, are capable of defining and identifying translocations that may be so close to the chromosomal termini as to be otherwise cryptic.

Both types of probes may be used if desired. Sources of such probes are available from the American Type Culture Collection, and similar depositories.

The oligonucleotide probes used in accordance with the methods of the present invention are of a size sufficient to permit probe penetration and to optimize reannealing hybridization. In general, labeled DNA fragments smaller than 500 nucleotides in length, and more preferably of approximately 150-250 nucleotides in length, probes are employed. Probes of such length can be made by synthetic or semi-synthetic means, or can be obtained from longer polynucleotides using restriction endonucleases or other techniques suitable for fragmenting DNA molecules. Altematively, longer probes (such as polynucleotides) may be employed.

Most preferably, the oligonucleotide probes are synthesized so as to contain biotinylated or otherwise modified nucleotide residues. Methods for accomplishing such biotinylation or modification are described in U.S. Pat. Nos. 4,687,732; 4,711,955; 5,328,824; and U.S. Pat. No. 5,449,767, each herein incorporated by reference. Biotinylated nucleotides and probes are obtainable from Enzo Biochem, Boehringer Mannheim, Amersham and other companies. In brief, such biotinylated or otherwise modified nucleotides are produced by reacting a nucleoside or nucleotide with a mercuric salt under conditions sufficient to form a mercurated nucleoside or nucleotide derivative. The mercurated product is then reacted in the presence of a palladium catalyst with a moiety (e.g., a biotin group) having a reactive terminal group and comprising three or more carbon atoms. This reaction adds the moiety to the purine or pyrimidine ring of the nucleoside or nucleotide.

In a highly preferred embodiment, such modified probes are used in conjunction with competitor DNA in the manner described by Ward et al. (WO90/05789), herein incorporated by reference. Competitor DNA is DNA that acts to suppress hybridization signals from ubiquitous repeated sequences present in human and other mammalian DNAs. In the case of human DNA, alu or kpn fragments can be employed, as described by Ward et al. (WO90/05789). Initially, probe DNA bearing a detectable label and competitor DNA are combined under conditions sufficient to permit hybridization to occur between molecules having complementary sequences. As used herein, two sequences are said to be able to hybridize to one another if they are complementary and are thus capable of forming a stable anti-parallel double-stranded nucleic acid structure. Conditions of nucleic acid hybridization suitable for forming such double stranded structures are described by Maniatis, T., et al. (In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y. (1982)), by Haymes, B. D., et al. (In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985), and by Ried, T. et al. (Proc. Natl. Acad. Sci. (U.S.A.) 89:1388-1392 (1992)). For the purpose of the present invention, the sequences need not exhibit precise complementarity, but need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure. Thus, departures from complete complementarity are permissible, so long as such departures are not sufficient to completely preclude hybridization and formation of a double-stranded structure.

The quantity of probe DNA combined with competitor DNA is adjusted to reflect the relative DNA content of the chromosome target. For example, as disclosed by Ward et al. (WO90/05789), chromosome 1 contains approximately 5.3 times as much DNA as is present in chromosome 21. Thus, a proportionally higher probe concentration would be employed when using chromosome 1 specific probes.

The resulting hybridization mixture is then treated (e.g., by heating) to denature the DNA present and is incubated at approximately 37° C. for a time sufficient to promote partial reannealing. The sample containing chromosomal DNA to be identified is also heated to render it susceptible to being hybridized to the probe. The hybridization mixture and the sample are then combined, under conditions sufficient to permit hybridization to occur. Thereafter, the detection and analysis of the hybridized product is conducted by detecting the fluorophore label of the probe in any of the methods described below.

In an alternative embodiment, a modification of the method of Ried T. et al. (Proc. Natl. Acad. Sci. (U.S.A.) 89:1388-1392 (1992), herein incorporated by reference) is employed. Thus, probes are labeled either directly (e.g., with fluorescein) or indirectly (e.g., with biotinylated nucleotides or other types of labels), and permitted to hybridize to chromosomal DNA. After hybridization, the hybridized complexes are incubated in the presence of streptavidin, that had been conjugated to one or more fluors. The streptavidin binds to the biotinylated probe of the hybridized complex thereby permitting detection of the complex, as described below.

C. The Preferred Fluorophores of the Invention

By labeling with two or more fluors in combination, it is possible to discriminate between many more objects than there are available fluors. The simplest way to implement such labeling is by Boolean combination, i.e., a fluor is either completely absent (0) or present in unit amount (1). For a single fluor A, there is only one useful combination (A=1). For two fluors A, B there are 3 useful combinations (A=1, B=0; A=0, B=1; A=1, B=1). For three fluors A, B, C, there are 7 combinations (A=1, B=0, C=0; A=0, B=1, C=0; A=0, B=0, C=1; A=1, B=1, C=0; A=1, B=0, C=1; A=0, B=1, C=1A=1, B=1, C=1). There are 15 combinations of 4 fluors, 31 combinations of 5 fluors, 63 combinations of 6 fluors, and so on.

To uniquely code all 24 chromosome types in the human genome, 5 distinguishable combinatorial fluors are needed. With a 5-fluor set, 15 chromosomes can be distinguished using combinations of 4 of the 5 fluors. The labeling of the remaining 9 chromosomes requires all five fluors to be used combinatorially. Seven of the available 5-fluor combinations are not required. Thus, there is a certain amount of latitude available to avoid any 5-fluor combination that might prove particularly hard to resolve. In particular, quaternary or quinternary combinations may be avoided.

One aspect of the present invention concerns the identification of a set of seven fluors that are be well resolvable by the excitation-emission contrast (EEC) method.

As indicated above, multi-fluor combinatorial labeling depends in general on acquiring and analyzing the spectral signature of each object i.e., obtaining the relative weighting coefficients of the component fluors. Because full spectroscopic analysis of mixed fluor spectra (e.g., by interferometry) is not yet sufficiently developed, the method chosen was conventional bandwidth-restricted widefield imaging using epi-fluorescence triplets, viz. excitation filter, dichroic reflector and emission bandpass filter. The limited spectral bandwidth available for imaging (roughly 380-750 nm), and the extensive overlap between the spectra of organic fluors, makes separating multiple fluors spectroscopically during the imaging step a significant technical challenge.

To make software segmentation of the source images as straightforward as practicably possible, a target figure of <10% crosstalk between any given fluor and the two adjacent channels was set. Computer modeling indicated that for DAPI plus the five combinatorial fluors FITC, Cy3, Cy3.5, Cy5, Cy5.5, this level of contrast cannot be attained using either excitation selection or emission selection alone, no matter how narrow the filter bandwidths. Thus, both excitation selection and emission selection must be invoked simultaneously. This is referred to as excitation-emission contrast (EEC).

Contrast ratio plots were first computed for each of the fluors vs. its two neighbors. These plots indicate regions where pairwise contrast is high enough to be useful. A constraint on the practically attainable contrast is that regions of high contrast generally lie far down the flanks of at least one of the spectra i.e., where excitation and/or emission are strongly sub-optimum. Further, to attain the required degree of selectivity it is necessary to use filters of bandwidths in the range 5-15 nm (cf. approx. 50 nm for ‘standard’ filter sets). Together, these impose a severe sensitivity penalty. The goal of 10% maximum crosstalk represents an acceptable, practical compromise between sensitivity and selectivity.

A fundamental asymmetry exists between excitation contrast and emission contrast. For low-noise detectors such as the cooled CCD, restricting the excitation bandwidth has little effect on attainable image S/N ratio; the only penalty is the need for longer exposure times. Restricting the emission bandwidth is very undesirable, however, since every fluorescence photon blocked by the filter represents irreversible photochemical bleaching of the fluor. For this reason, the highest practicable contrast was invoked on the excitation side. A suitable emission filter was then found to give the necessary EEC ratios. Filter selection is additionally constrained by the fact that each channel must adequately reject both adjacent channels simultaneously: improving one may significantly degrade the other. Good contrast was attainable in practice for all fluors except the Cy5.5/Cy5 pair, which is marginal. For this reason, Cy7 was later substituted for Cy5.5. Other considerations relating to choice of filters include:

1. Commercial narrow-band interference filters may have a large amount of wedging i.e., non-parallelism between the top and bottom faces. This results in large image shifts (up to several microns equivalent). The shift is a vector characteristic of each filter and its orientation in the epicube. Thus, automatic compensation for image displacement is a necessary part of the processing software.

2. Manufacturing variations of a few nm in peak wavelength and FWHM specifications can have significant effects on the EEC ratios. Filter errors to long wavelength may be fine-tuned by tilting, but this option is severely curtailed in the case of emission filters because of increased image aberrations and worsened pixel shifts. There is no equivalent way to compensate short-wavelength errors.

3. the need to prevent infra-red light emitted by the arc lamp from reaching the detector. Silicon CCD's are extremely sensitive in this region. Filter sets for the blue and midvisible fluors were found not to need additional IR blocking, but loss of image contrast due to spurious IR was found to be a serious problem for the red—far red—near IR fluors. Heat filters routinely used in microscopy (e.g., Schott BG-38 glass) are completely inadequate to alleviate this problem. Thus, extensive additional blocking was required. However, available commercial interference filters for infra-red blocking filters also transmit poorly in the near UV, and thus cannot be inserted in the excitation path. Instead, it was found necessary to put the IR blocking filters into the emission path. To minimize loss of image quality by insertion of these filters in the image path, they are placed inside the CCD camera, immediately in front of the window. In practice, two interchangeable filters were chosen, one for use with Cy5, Cy5.5 (Oriel #58893; 740 nm cutoff) and one for use with Cy7 (Oriel 58895; 790 nm cutoff).

The first member of the set of fluors is the counterstain DAPI, which gives a weak G-like banding pattern. Five of the remaining six fluors may be used combinatorially to paint the entire human chromosome set. All are available as avidin conjugates (for secondary detection of biotinylated probe libraries) or directly linked to dUTP (for direct labeling).

Thus, a set of six fluors and corresponding optical filters spaced across the spectral interval 350-750 nm was identified that achieve a high discrimination between all possible fluor pairs. These fluors comprise the preferred fluors of the present invention and are: 4′-6-diamidino 2-phenyl indole (DAPI), fluorescein (FITC), and the new generation cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. Of these Cy3, Cy3.5, Cy5 and Cy7 are particularly preferred, The absorption and emission maxima for the respective fluors are: DAPI (Absorption maximum: 350 nm; Emission maximum: 456 nm), FITC (Absorption maximum: 490 nm; Emission maximum: 520 nm), Cy3 (Absorption maximum: 554 nm; Emission maximum: 568 nm), Cy3.5 (Absorption maximum: 581 nm; Emission maximum: 588 nm), Cy5 (Absorption maximum: 652 nm; Emission maximum: 672 nm), Cy7 (Absorption maximum: 755 nm; Emission maximum: 778 nm), Complete properties of selected fluorescent labeling reagents are provided by Waggoner, A. (Methods in Enzymology 246:362-373 (1995) herein incorporated by reference). In light of the above, it is readily apparent that other fluorophores and filter combinations having adequate spectral resolution can alternatively be employed in accordance with the methods of the present invention.

D. Methods for the Detection of Fluorescent In situ Hybridization

1. The Theory of Fluorescence Detection

Of the various methods for contrast generation in site-specific labeling, fluorescence is arguably the most powerful, because of its high absolute sensitivity and multiparameter discrimination capability. Modem electronic cameras used in combination with high numerical aperture microscope objectives and state of the art optical filters are capable of imaging structures labeled with as little as a 10-100 fluor molecules per pixel. Thus, fluor-tagged single-copy DNA sequences as small as a few hundred bases in size are detectable under favorable conditions. The availability of families of spectrally distinguishable fluors makes simultaneous imaging of several different targets in the same specimen possible, either directly or through combinatorial or analog multiplex methods. In principle, multi-fluor discrimination may be based on differential excitation of the fluors, differential emission, fluorescence lifetime differences, or on more complex but still analyzable observables such as fluorescence anisotropy. This discussion assumes an epi-imaging geometry. Table 1 describes the symbols and operators relevant to the theoretical considerations of fluorescence.

TABLE 1 Units Definition Instrument Parameter ψ_(s)(λ) photon s⁻¹ nm⁻¹ spectral distribution of source, assumed to be an isotropic radiator over 4π steradians φ_(s) dimensionless collection of efficiency of condenser optics ψ1(λ) photon s⁻¹ m⁻² nm⁻¹ spectral distribution of photons in collimated beam of excitation light impinging on excitation filter F1 f1(λ) dimensionless transmittance function of excitation filter F1 ψ2(λ) photon s⁻¹ cm⁻² nm⁻¹ spectral distribution of photons in collimated beam of excitation light emerging from F1 and impinging on dichroic beamsplitter DB1 R(λ) dimensionless reflectance function of dichroic beamsplitter DB1 T(λ) dimensionless transmittance function of dichroic beamsplitter DB1 ψ3(λ) photon s⁻¹ cm⁻² nm⁻¹ spectral distribution of photons in collimated beam of fluorescence emerging from DB1 and impinging on emission filter F2 f2(λ) dimensionless transmittance function of emission filter F2 ψ4(λ) photon s⁻¹ cm⁻² nm⁻¹ spectral distribution of photons in collimated beam of excitation light emerging from F2 and entering the entrance pupil of the objective lens μ dimensionless linear magnification factor of objective lens ψ5(λ) photon s⁻¹ cm⁻² nm⁻¹ spectral distribution of photons in focused beam of excitation light traversing the specimen plane φ_(d)(λ) dimensionless quantum efficiency of detector Ω pixel.μ⁻¹ magnification factor of final image at detector Fluor Parameter ε(λ) M⁻¹ cm⁻¹ molar decadic extinction coefficient of fluor F_(a). τ s decay lifetime of excited state of fluor F_(a). σ_(Fa)(λ) cm⁻¹ photon absorption cross-section of fluor F_(a). φFa dimensionless fluorescence quantum efficiency of fluor F_(a). ƒ_(a)(λ) dimensionless normalized spectral distribution of fluorescence from fluor F_(a).

Whether a given excitation rate at pixel location p will give an acceptable fluorescence signal/noise ratio (defined as S/N=[signal mean]/[variance due to all noise sources]) in a given integration period depends on the number of fluor molecules within p, their quantum yield and photochemical stability, and the quantum efficiency and noise performance of the detector.

In the limit of weak excitation (ψ^((λ)).σ_(F) ^((λ))<<τ⁻¹), the rate of excitation of the N molecules of fluor F within pixel p in object space is:

R(p)=N∫ _(i)ψ^(5(λ,p)).σ_(F) ^((λ)) .dλs ⁻¹

ψ^(5(λ)) is the spectral distribution of exciting light passing through the focal plane of the microscope (photon nm⁻¹.cm⁻².s⁻¹). It is approximately given by ψ_(s) ^((λ)).φ_(s).f1(λ).R(λ).α².μ, where α=diameter of objective lens entry pupil/diameter of collimated beam from condenser. The integral is taken over the bandwidth (i) for which the fluor has non-zero absorption cross section σ_(F) ^((λ)), which is related to the molar decadic extinction coefficient ε_(F)(λ) by the expression:

σ_(F) ^((λ))=3.825×10⁻²¹ε_(F) ^((λ)) cm².

In practice, ∫_(i)ψ^(5(λ,p)). dλ can be measured with a bolometric detector such as a calibrated micro-thernopile placed at or near the focal plane of the objective lens.

For a perfectly noiseless detector and a non-bleachable fluor, the S/N of each pixel increases indefinitely as (photons detected)^(1/2). How rapidly S/N increases depends on excitation strength, but the relationship between S/N and dose does not. The effect of non-zero bleaching constant is to change the t^(1/2) function to an asymptotic function, the form of which depends on the bleaching mechanism. However, because the bleaching rate and the signal strength are linearly related, the asymptote once again does not depend at all on excitation rate, although the speed of approach to the asymptote does. If finite camera noise is added to photobleaching, it is found that the S/N climbs to a maximum value, then falls as the fluor is exhausted. Now both the kinetics and the peak S/N depend of excitation rate, in general the faster the excitation the higher the maximum attainable S/N. However, for contemporary cooled CCD cameras the dark noise is so low that it can be virtually ignored on the timescale of bleaching (typically a few minutes); a more important factor in determining ultimate S/N is the stray light background (esp. from nonspecific luminescences and leakage of excitation light).

Note that although the microscope objective compresses the excitation beam, in a nonconfocal microscope it does not focus it to a point and so has no bearing on the fluorescence image resolving power (incoherent emitter).

The commonest excitation source for fluorescence microscopy is the high pressure short mercury arc, whose spectrum consists of pressure-broadened lines from the UV to the middle region of the visible spectrum (principal wavelengths are 334.1 nm, 365.6 nm, 404.7 mm, 435.8 nm, 546.1 nm. 577.9 nm), superimposed on a weaker thermal continuum. Many fluorophores have excitation spectra that overlap one or other of the mercury lines to an acceptable extent. Others (of which the best known is FITC) do not, but may be adequately excited by the continuum if a wide enough excitation bandwidth is employed. Another common source is the high pressure xenon short arc, which produces an almost uniform continuum from ca. 300 nm: to beyond 900 nm. However, the power nm⁻¹, is almost everywhere less than the mercury continuum for the same arc wattage. If high intensity light with no structure is required (e.g., for fluorescence ratio imaging) a high CW power or pulsed quartz halogen lamp outperforms the xenon beyond about 450 nm. Certain fluors are well matched to laser excitation (e.g., Ar+@488 nm for FITC, He—Ne @632.8 nm for Cy5, semiconductor diode—pumped YAG @680 nm for Cy5.5).

In single-fluor imaging, use of the available spectral bandwidth is rarely stringent. The excitation filter F1 and dichroic beamsplitter DBI can usually be chosen to give adequate overlap between the source spectrum and the fluor excitation spectrum. If an arc line is available, the F1 bandpass need be no wider than the line. If not, and part of the thermal continuum must be used, the wider the F1 bandpass the greater will be the available excitation flux. However, with low noise integrating detectors the goal of high excitation efficiency is generally secondary to the need to exclude excitation light from the emission path. This limits how close the excitation and emission bandpasses can be placed to one another, and hence constrains the excitation bandwidth. For fluors with small Stokes' shifts, high quality filters with very steep skirts are required. The excitation filter must be rigorously ‘blocked’ on the long wavelength side, and have no pinholes, scratches, or light leaks around the edge.

Dichroic beamsplitters are currently much less ‘evolved’ than bandpass interference filters, meaning that the slopes of their transmit <−> reflect transitions are far less than the skirt slopes of premium notch filters, and there may be large spectral intervals where they oscillate between intermediate states of partial reflectance and transmittance. The main purpose of a dichroic beamsplitter is to improve the combined efficiency of excitation and emission, rather than to define the wavelength response of the instrument.

The resolvability of overlapping fluors in imaging microscopy may depend critically on the degree of excitation contrast that can be achieved (see C, below). The variation with wavelength of the ratio of the extinction coefficient of two fluors is the excitation contrast spectrum. It can readily be calculated from the digitized absorption spectra. Depending on the overlap of the absorptions, their ratio spectrum may either show a distinct peak or may grow indefinitely large. In either case, it is usually possible to choose an excitation wavelength that favors one fluor over another to a useful extent (from a factor of 3-4 fold up to a hundredfold or more). Some difficulties in obtaining high contrast multi-fluor images include:

a. The excitation wavelengths required for high contrast imaging are often far from the absorbance peaks. Thus, there may be a high degree of intensity trade-off to obtain high signal contrast vs. other fluors.

b. From the above, standard filter sets cannot be used.

c. Arc source spectral lines that are useful for exciting single fluors may not give high contrast discrimination against adjacent fluors.

d. When using a broadband source or the continuum spectrum of an arc source, the need for narrow excitation bandwidth may reduce the excitation flux to problematically low levels. It is sometimes possible to relax the constraints on excitation wavelength for the sake of more efficient excitation.

Collecting the fluorescence of F and imaging it onto the detector with high efficiency is the principal design goal of the emission optics. Operationally, it is even more important to have an efficient emission path than an efficient excitation path. The reason is that inefficient excitation causes inefficient photobleaching; the only penalty is a long image integration time (assuming a low-noise detector). On the other hand, any fluorescence lost on its way to the detector represents photobleaching without concomitant increase in the information content of the image.

The detector pixel p accumulates signal (detected photons) at a rate:

F ₍ p)=G. R(p).Φ_(F).∫_(ii) T(λ).f2(λ).φ_(d)(λ).dλ.s ⁻¹

The integral is over the bandwidth (ii) for which the detector quantum efficiency φ_(d)(λ)>0. G represents the efficiency with which the optics gather the fluorescence and transmit it to the detector; it may be assumed to be wavelength independent to first order. The principal factor in G is the numerical aperture (NA) of the objective lens, which determines the fraction of the isotropically radiated fluorescence collected by the imaging system (§=1/π.sin⁻¹(θ)=1/π.sin⁻¹(NA/n), where θ is the half-angle subtended by the objective lens from its focal point. For an oil immersion lens with NA=1.3; n=1.515; §=0.328). NA additionally determines the spatial resolving power of the microscope, because it scales the dimensions of the Frauenhofer diffraction pattern produced in the image plane by a point source in the specimen plane. Several ‘rules’ are in use for specifying the resolving power of a lens, depending on how much overlap of the Airy discs of two adjacent objects is deemed to constitute the threshold of resolution. The commonly used Rayleigh criterion is r=1.22λ/2NA (e.g., for the above NA=1.3 lens working at 500 nm, r=0.24 μ).

For a noise-free detector, image ‘noise’ at p is determined by the statistical variance in the number S(p) of fluorescent photons detected in time interval Δt. The only detector characteristic that has any bearing on this is its quantum efficiency φd(λ).

In the absence of photobleaching (probability of destruction per hit Π_(b)=0), S(p)=F(p).Δt, with variance S(p)^(1/2), i.e., S/N=S(p)^(1/2). Image quality will therefore increase indefinitely with Δt, though at an ever decreasing rate.

In the presence of photobleaching (Π_(b)≠0), S(p) is an integral of the form:

S(p)=F(p)∫_(Δt) f(t).dt

where f(t) is the photobleaching decay function. S/N rises along a more or less complex path to an asymptotic value that corresponds to total exhaustion of the fluor. For the case of a unimolecular photobleaching process this would be an exponential function. i.e., N(p,t)=N₀(1−-exp-[k_(b)/Σk].t) where k_(b)=Π_(b) ⁻¹ is the photobleaching constant and Σk represents all other processes by which the excited state of F is deactivated. Note that the normalized asymptote in this first-order system depends only on k_(b)/Σk, and is independent of the strength of the excitation. Thus, the extent of bleaching is exponentially related to the accumulated excitation dose, but is independent of the path. In reality, however, bleaching of fluors in solution may be mechanistically and kinetically much more complex. A common mechanism involves ring opening following peroxidation of the fluor excited state, e.g., by ¹O₂ or O₂ ²⁻. This type of bleaching may be considerably slowed by rigorous deoxygenation or by the use of oxygen radical scavengers (i.e., antifade agents) such as tertiary amines (p-phenylene diamine or DABCO). Nevertheless, other (as yet poorly characterized) irreversible processes are not excluded, including reactions with impurities.

A nonideal detector contributes noise of many kinds, detailed analysis of which may be intractable. The simplest noise component is fluctuation in the so-called ‘dark current,’ i.e., the flow of thermally excited carriers within the detector. If this noise is assumed to be random, it adds to the photon shot noise in RMS fashion. Thus, if the mean photogenerated signal is F s⁻¹ and the mean dark count is D s⁻¹, the S/N after time Δt is F.(Δt)^(1/2)/(F+D)^(1/2); S/N still increases as (Δt)^(1/2), but more slowly than for a noiseless detector. When photobleaching is present, however, the situation is entirely different. In this case, S/N rises initially as (Δt)^(1/2), but at some point reaches a shallow maximum and then begins to fall again. as the fluorescent signal declines but the thermal noise power remains constant. In this case, it is in principle desirable to continuously monitor the S/N of the image, and terminate the exposure when the peak is reached. Most commercial digital imaging systems make no provision for this. Fortunately, state of the art cooled CCD cameras have so little dark noise per pixel (typically<0.01 electron s⁻¹ in inverted clock mode) that S/N would not begin to fall until almost complete exhaustion of the fluor. In practice, autofluorescences and stray light dominate system performance long before the noise threshold of the CCD is reached.

The principal design goals for a single-fluor imaging system are:

1. To achieve an adequate rate of excitation of the fluor (F).

2. To collect the fluorescence of F and image it onto the detector with high efficiency and with the necessary spatial resolution.

3. To prevent reflected and/or scattered excitation light from reaching the detector.

Design of the emission channel for a single fluor is straightforward. The dichroic beamsplitter transition wavelength is specified at for example, 20 mn to the red of the excitation passband. This ensures a high level of rejection of exciting light reflected and/or scattered from the specimen and/or microscope optics. The emission filter cut-on is usually considerably steeper than the dichroic edge, and so can be placed practically coincident with it. The most efficient emission filter is a long-pass element. The preferred filter of this type is Schott glass, which transmits upwards of 90% of all fluorescence to the red of its cut-on, while rejecting other light (especially any excitation light that gets through the dichroic beamsplitter) to very high order—typically>10⁵. However, it is usually inadvisable to leave the emission channel ‘wide open’ into the near infrared, especially with silicon detectors which have high sensitivity there.

The multiparametric imaging of the present invention not only increases the throughput of information about the system under observation and makes more efficient use of the biological material, but also can reveal spatial and temporal correlations that might otherwise be difficult to establish reliably. When a large number of different objects must be visualized, two or more labels can be used combinatorially, which permits discrimination between many more object types than there are spectrally distinguishable labels. Some examples of multi-fluor imaging are:

a. The co-distribution of proteins in structures such as nicrotubule networks may readily be visualized using immunolabels linked to different fluors.

b. Multiple genes may be simultaneously mapped by fluorescence in situ hybridization (FISH) to a single metaphase chromosome spread. Such signals cannot usually be discriminated reliably on the basis of intensity alone, and are usually morphologically identical (diffraction-limited points). However, they are readily discriminated by discrete or combinatorial multi-fluor labeling.

c. Identification of small chromosomal translocations is most readily done by painting with chromosome specific DNA probe libraries linked to separable fluors, used either singly or combinatorially.

d. Analysis of mixed populations of morphologically identical bacteria can also be achieved using species-specific DNA or ribosomal RNA probes coupled to separable fluors.

The primary design goal of a multi-fluor imager (in addition to those for of a single fluor imager) is to spectrally resolve the fluorescence at any pixel location into components corresponding to each fluor. Methods for spectrally resolving complex signals in fluorescent microscope images are outlined below.

There are several ways to resolve spectrally complex signals, i.e., to determine which fluors contribute to the fluorescence at a given pixel location. The most general method in principle is to spectrally disperse the 2D image along a third axis, orthogonal to the x,y axes. This amounts to imaging through an optical system with a very large amount of chromatic aberration, such that at each position on the z axis there is an image x,y that contains only a small spectral bandwidth λ+Δλ. An area detector with very small depth of field (such as a spatially filtered confocal imager) could then be moved incrementally along the z axis to obtain a family of images, each containing its own small spectral interval. A trace through the images at given x,y would constitute an emission spectrum for that pixel. Unfortunately, implementing such a scheme is technically very difficult.

If the spectrum of only a small number of objects within the image is required (such as individual stars in a telescope image), a solution is to extract the light corresponding to each object with a probe (e.g., a fiber optic) and disperse it with an imaging spectrograph onto a 1-dimensional array detector. To be useful in microscopy, such a device would have to be arbitrarily positionable in the field, and have an adjustable acceptance area.

The most reasonable method for full spectral analysis in microscopy is to image through a variable narrow band filter. An image is recorded at each wavelength; intensity values at a given pixel location through the series represent a weighted emission spectrum that can be fit to a linear combination of the known spectra of the component fluors. The coefficients are products of the relative molar amounts of the fluors with their extinction coefficients at the exciting wavelength and their fluorescence quantum yields. If the last two are known, the first is obtainable from the fit. In general, it is necessary to take several such image sets, at several excitation wavelengths, to get a unique fit. With enough iterations, this process generates a 3D surface of intensity values as a function of both excitation and emission wavelength. This comprises a complete spectral signature of the pixel, giving a very highly constrained solution for the relative amounts of its component fluors. The mole ratios could be mapped back onto the x,y coordinates of the image, with appropriate pseudocolor coding, to give a ‘composition map’. This general (and quantitative) method has a number of technical difficulties, although none of them is insurmountable. The first is that a large number of images are required to evaluate a single microscope field. Imaging time is long, and extensive differential photobleaching of the fluors would make it impossible to achieve a self consistent “fit” to the spectral data. Instrument stability is also an issue, particularly with arc sources, the output spectra of which change throughout their life. Finally, the amount of computation required to generate a composition map would realistically limit the analysis to small image regions only.

For most applications, there is no need of a full-blown spectral analysis capability because the fluors to be analyzed are known ahead of time. Thus, it is only necessary to be able to identify which fluor is which, and, for multiplex imaging, to ascertain the relative amount of each present. A preferred approach to this category of problems involves the use filter sets that achieve a high degree of selective excitation and visualization of each component fluor. The ideal system of this type would perfectly isolate each fluorophore, i.e., “one image—one fluor” and all off-diagonal elements in the matrix of intensity coefficients would be zero. However, the facts that the spectra of typical fluorophores are 10-30% of the total available bandwidth, and emission filters must have significant bandwidths to pass usable amounts of light place severe limitations on the attainable degree of spectral isolation. Nevertheless, it is not difficult to achieve useful levels of contrast between suitably chosen fluors, such that residual crosstalk can be removed numerically.

The excitation-emission contrast (EEC) approach is in principle applicable to analysis of images involving multiple fluors with fine-grained distributions of mole fraction (e.g., fluorescence ratio imaging), subject to image S/N and the limitations of differential bleaching rates and source instability. However, it is particularly simple for the limiting case of combinatorial labeling, in which fluors are multiplexed in a strictly Boolean fashion (present=1, or absent=0). In this case, it is necessary only to be able to reliably discriminate between a 1 and a 0, for which a 10:1 intensity ratio between any fluor and its neighbors would be sufficient. However, for many useful fluors this is not achievable on the basis of excitation contrast or emission contrast alone. Simultaneous selection on the basis of both excitation and emission are required.

In some circumstances it is required to excite and image several fluors simultaneously, e.g., for direct viewing and/or color video recording. Excitation and emission optics that have no wavelength selectivity cannot be used, because the excitation light scattered into the detector would overwhelm the fluorescence by several orders of magnitude. One solution is to use multiple-bandpass filters designed for the specific set of fluors to be used. The excitation filter defines narrow passbands that overlap the fluor excitation spectra. The emission filter defines similar passbands that interdigitate between the excitation bands and overlap the fluor emission spectra (the reddest fluor could use a long-pass filter). The dichroic beamsplitter alternates between reflect (overlapping the excitation passbands) and transmit (overlapping the emission passbands).

Use of a multi-pass filter set also has an advantage even when multicolor visualization is not required, viz. the absence of geometric displacement (pixel shifts) between signals passing through the several emission passbands. However, when used with a greyscale imaging system, it is necessary to make either the excitation filter or the emission filter switchable, in order to determine which greyscale signal corresponds to which fluor. The preferred choice is to switch the excitation filter, because this causes no image displacement.

A second theoretical solution would be to excite at a wavelength where all the fluors absorb. This is often possible because many fluors are excitable to states higher than S1 using photons in the middle UV, but because of internal relaxation processes give ‘normal’ fluorescence. For example, many laser dyes can be excited at the nitrogen laser wavelength, 337 nm, far to the blue of their visible absorbances. It would be straightforward to block such exciting light from the emission path, using a long-pass filter (e.g., 380 nm), while allowing all fluorescences to simultaneously reach the detector. Drawbacks to the use of UV excitation include increased rates of photochemical decomposition of the fluor, and the expense of suitable UV optics. Thus, the method has not found widespread use.

The multi-bandpass method has the limitation that construction of multiple bandpass elements giving adequate contrast between more than 3 fluors is extremely difficult. A generally more powerful approach is to construct optimized filter sets for each fluor, and switch them as needed. In the case of a single fluor, the primary goal on the excitation side is high excitation flux, to give a bright image. When imaging multiple fluors, however, this becomes secondary to the goal of achieving high contrast between fluors. In the weak overlap limit, fluors may be imaged by sequentially switching filters that are designed using the same criteria as single-fluor sets (except that long-pass emission filters are proscribed for all but the longest-wavelength fluor). Crosstalk of a few percent is usually allowable, and can be compensated numerically if necessary. More generally, though, generation of adequate contrast between fluors requires both strongly selective excitation and emission. The optimum wavelengths can be found by ratioing the spectra of adjacent fluors. These wavelengths may be far from the excitation maxima, implying a significant tradeoff between signal brightness and contrast. Standard filter sets cannot be used. The need for precisely placed excitation makes it less likely that a strong excitation line will be available, and also reduces the flux available from a continuum source. It may be necessary to pulse the broadband source (arc or filament lamp) to transiently very high output levels, or to supplement it with laser sources.

The principal technical problem with serial imaging is image displacement when filters are changed i.e., the coordinate systems of the individual members of an image series are not in precise registration. This problem arises from nonidealities in the emission channel optics. Two displacement components can be identified:

i. a reproducible offset that.is unique to each filter set. This component is a fixed vector, and arises mainly from imperfect parallelism (i.e., wedging) between the top and bottom faces of the emission bandpass filter. There is also a small component due to wedging in the dichroic beamsplitter, but since this element is very thin the effect is minor. Since the wedging vector is a constant for each filter set, it can be automatically removed in the computer. The size of the offset can also be reduced to very small values (<0.1 μ) by selecting emission filters for a high degree of parallelism e.g., by measurement in a laser autocollimator.

ii. a random component due to vibration and hysteresis in the filter switching mechanism. The magnitude of the noise depends on the filter switching mechanism. The worst are manual push-pull sliders, particularly when the operator actuates them using uncompensated forces. The best are motorized filter cassettes, in which all mechanical torques act against the microscope body.

Note that both the constant and random components of the image offset noise are minimized by using an objective lens of the highest possible magnification, and the minimum amount of magnification in the camera projection optics. Image displacements encountered in optical microscopy are linear vectors only; there is no evidence for rotation or significant changes in scale (magnification).

Design considerations for high contrast imaging of multiple overlapping fluors are similar to those already outlined for the excitation channel. By calculating the ratio of each fluor's emission spectrum to that of its neighbors, it is possible to identify spectral regions of high contrast that can be defined with narrowband filters. High contrast is often achievable only at the cost of throwing away significant amounts of the fluorescence (90% or more). Inefficiency in the fluorescence channel is much more damaging to image S/N than excitation Inefficiency. Thus, where possible, selective excitation is the preferred method of achieving contrast between multiple fluors. In difficult cases, however, both excitation and emission contrast are required.

2. Optical Filter

As indicated, in a preferred embodiment of the invention, the detection of fluor is accomplished using optical filters, in a modification of the method of Ried, T. et al. (Proc. Natl. Acad. Sci. (U.S.A.) 89:1388-1392 (1992), herein incorporated by reference).

Imaging DAPI

4′,6-diamidino-2-phenylindole (DAPI) is a commonly used DNA counterstain that intercalates preferentially into AT-rich regions of chromosomes and so gives rise to a weak G-type banding pattern. It is a very bright fluor (ε=3.3×10⁴ M⁻¹ cm⁻¹ at 347 nm, with approx. 20-fold enhancement of fluorescence quantum yield when intercalated into the minor groove of double-stranded DNA), and is relatively resistant to photobleaching. The following points are relevant to imaging DAPI with high contrast against its neighbors:

a. DAPI has very broad excitation and emission spectra, and a very large Stoke's shift. Thus, although the DAPI excitation maximum (347 nm) is to the blue of Cascade Blue (CB), the fluorescence of DAPI peaks to the red of Cascade Blue, and actually overlaps quite strongly with FITC.

b. The usual excitation wavelength for DAPI (Hg 366 nm line) cannot be used in this application, because it is almost isosbestic for DAPI and CB and thus gives no excitation contrast.

c. The peak of the DAPI/CB excitation contrast spectrum is at 320 nm, which is too far into the UV to pass through the microscope optics. An acceptable compromise is to excite with the Hg 334.1 nm line, which the microscope transmits with about 30% efficiency. The Ealing 35-2989 interference filter has an appropriate bandpass. The excitation contrast ratio for DAPI/CB at 334.1 nm has an absolute value of 4.0.

d. To further increase the DAPICB selectivity, emission contrast must be used in addition to excitation contrast. Note that DAPI emits the bulk of its fluorescence to the red of CB. The wavelength of maximum emission contrast for DAPI vs. both CB and FITC is 490 nm. A suitable imaging-grade filter is the Omega 485DF22. There are no mercury lines within its bandpass, so good DAPI images with low flare are expected. The calculated emission contrast between DAPI and both Cascade Blue and FITC is 6.8. Hence, the overall contrast achievable for DAPI vs. Cascade Blue is approximately 27-fold. The overall contrast of DAPI vs. FITC is very much higher than this, because at the DAPI excitation wavelength the excitation of FITC is close to zero.

e. The Omega 450DRLP02 dichroic beamsplitter is very well matched to the proposed excitation and emission filters.

Imaging Cascade Blue

Cascade Blue (CB) has a broad, two-peak excitation spectrum that overlaps DAPI extensively, though not its neighbor on the red side (FITC). The Stokes shift for CB is very small, i.e., there is very extensive overlap between its excitation and emission spectra. These factors combine to make imaging CB in the presence of DAPI problematical. To sumrnmarize:

a. The peak of the CB/DAPI excitation contrast spectrum is at 396-404 nm. However, because of the very small Stokes' shift of CB this is very close to the emission contrast peak (408 nm). Since it is more important for image S/N to collect the emission with high efficiency than to excite with high efficiency, the 400-410 rim region is reserved for the CB fluorescence. The best compromise for CB excitation is the Omega 38OHT15, which overlaps with the Hg 366 nm line enough to provide good excitation strength.

b. To achieve any emission contrast at all vs. DAPI, the emission filter must be narrow and very carefully placed. A suitable filter is the Omega 405DF10, but the theoretical maximum contrast is only 2.5. Thus, imaging CB while excluding DAPI is expected to be marginal.

c. Cascade Blue exhibits high excitation contrast vs. FITC (contrast ratio with the Omega 38OHT15 filter=6).

d. The emission contrast ratio for CB/FITC goes to very large values below 490 nm, and is essentially infinite at the position of the 405DF10 filter.

The above considerations indicate that it will be possible to use either DAPI or Cascade Blue, but not both together unless the DAPI counterstain is weak. Cascade Blue is well separable from FITC and the other five combinatorial fluors considered herein.

Imaging FITC

The following points are relevant to high-contrast imaging of FITC:

a. The excitation spectrum of FITC has insignificant overlap with that of Cascade Blue (contrast parameter Rb for FITC/CB becomes extremely large beyond 420 nm). This makes it possible to excite FITC on the high frequency shoulder of its absorbance, so avoiding appreciable excitation of Cy3.

b. There is no mercury line that overlaps well with the FITC excitation spectrum. Thus, it is necessary to use the continuum to excite this fluor, as with single-fluor imaging. The gap between FITC and CB makes it possible to use a very wide excitation filter; the Omega 455DF70 is well suited to this function.

c. The Omega 455DF70 bandpass also corresponds fortuitously to the maximum in the excitation contrast ratio for FITC/Cy3 (460 nm; absolute extinction ratio R_(a)=8.8).

d. The emission spectrum of FITC overlaps that of Cy3 (on the red side) and both DAPI and CB (on the blue side) to an appreciable extent. However, because the conditions defined for FITC excitation do not excite DAPI or Cascade Blue at all, they are not relevant. The FITC/Cy3 emission contrast ratio goes to very high values below 540 nm. Thus, the Omega 530DF30 filter gives very high emission contrast, which compounds the high excitation contrast ratio for FITC vs. Cy3 (8.8) given by the 455DF70. It therefore appears possible to image FITC very cleanly.

Imaging Cy3

The following points are relevant to high-contrast imaging of Cy3:

a. The absorbance peak of Cy3 is at 551 nm, at which wavelength the excitation of FITC is essentially zero (contrast parameter Rb jumps to extremely high values to the red of 525 nm).

b. The Cy3 extinction peak overlaps strongly with the Hg 546.1 nm line.

c. The excitation contrast ratio for Cy3/Cy3.5 is everywhere small, and varies weakly with wavelength. At 551 nm, the absolute value of the excitation contrast for Cy3 vs. Cy3.5 is less than 2, and it only rises significantly far to the blue where the Cy3 absorbance is very low and FITC absorbance is high. In fact, the apparent rise in R_(a) around 460-470 nm may be an artifact of the low absolute precision of the spectra in that region. From the above discussion, it is clear that the excitation contrast available for Cy3 vs. Cy3.5 is too low to be useful.

d. The emission contrast ratio for Cy3/Cy3.5 rises abruptly below 570 nm. At the 567 nm peak of the Cy3 fluorescence, the absolute value of S_(a) is approximately 6. This, combined with the factor 2 in the excitation contrast parameter, should just about meet the goal of a 10-fold discrimination between Cy3 and Cy3.5. The Ealing 35-3722 narrowband interference filter is suitable, although it does overlap the Hg 577/579 line significantly. Any stray light from this line getting through the 546DF10 filter is, however, expected to be well blocked by the chosen dichroic, Omega 560DRLP02.

e. The inability to differentially excite Cy3 vs. Cy3.5 means wasteful bleaching of Cy3.5 during imaging of Cy3.

Imaging Cy3.5

The following points summarize high contrast imaging with this dye:

a. The excitation contrast ratio between Cy3.5 and Cy3 rises to very high values beyond approximately 565 nm. This region includes the peak of the Cy3.5 excitation spectrum.

b. The mercury arc line at 577/579 nm is almost exactly coincident with the peak of the Cy3.5 absorbance. At this wavelength the excitation contrast ratio is approximately 25, i.e., very strong selective excitation of Cy3.5 relative to Cy3 is possible. An ideal filter for this purpose is the Ealing 35-3763. Note that the high contrast is mainly a consequence of the Hg line position, not the filter bandwidth.

c. At the Hg 577/579 excitation wavelength, the excitation contrast ratio for Cy3.5 relative to Cy5 is also quite large (absolute value approximately 8.0).

d. The emission contrast parameter for Cy3.5 vs. Cy3 is small at all wavelengths where the Cy3.5 emission is usefully strong, i.e., isolation of Cy3.5 from Cy3 must rely mainly on excitation contrast.

e. The emission contrast for Cy3.5 vs. Cy5 is also large over a considerable spectral interval (and rises to very high values below 640 nm). This permits a fairly broadband filter to be used to image Cy3.5; a suitable element is the Omega 615DF45. Almost no bleadthrough of either Cy3 or Cy5 into the Cy3.5 channel is expected with the above combination of excitation and emission conditions.

f. The Omega 590DRLP02 is a suitable dichroic for this channel.

Imaging Cy5

The following points are relevant to imaging with this dye:

a. Beyond 600 nm, the excitation contrast ratio for Cy5 vs. Cy3.5 gets very large, i.e., very high excitation contrast relative to Cy3.5 is possible.

b. The placement of the Cy5 excitation is determined by the extinction ratio relative to Cy5.5 rather than Cy3.5. The Cy5/Cy5.5 excitation contrast parameter peaks at 650 nm with a numerical value of 2.25. Thus, analogous with the Cy3/Cy3.5 pair, excitation contrast for Cy5/Cy5.5 is poor.

c. There is no Hg line available for exciting Cy5. Thus, with an arc source, the continuum must be used, analogously with FITC excitation. The “official” filter for exciting Cy5 in this way is the Omega 64ODF20, which will give an excitation contrast with Cy5 of about 1.8.

d. A much brighter source for exciting Cy5 is the He—Ne laser (632.8 nm). It does not, however, improve excitation contrast vs. Cy5.5.

e. The emission contrast for Cy5 vs. Cy3.5 peaks at 673 nm, just to the red of the fluorescence intensity peak. The closest available filter is the Omega 660DF32 where the emission contrast ratio is approximately 3.1. This compounds the very high excitation contrast for Cy5 vs. Cy3.

f. The emission contrast for Cy5 vs. Cy5.5 goes to very large values at wavelengths shorter than 675 nm. The Omega 660DF32 filter is ideally set up to take full advantage of this. Unfortunately, it is uncomfortably close to the 640DF20 exciter, so some flare from reflected/scattered excitation light is to be expected. Use of the He—Ne laser would remove this problem.

g. The best available dichroic beamsplitter for Cy5 imaging is the Omega 645DRLP02, particularly if a He—Ne is used as the excitation source.

Imaging Cy5.5

CY5.5 is the penultimate dye of the set, and is very well separated from Cy7. Thus, only its contrast relative to Cy5 need be considered in detail:

a. The contrast parameter R_(b) for the Cy5.5/Cy5 pair rises to large values to the red of 670 nm. Thus, it is possible to achieve very high excitation contrast for this pair of fluors (analogously to Cy3.5/Cy3).

b. As with Cy5, the Hg arc is a poor source for exciting Cy5.5. The best available source is a 680 nm diode-pumped frequency doubled YAG microlaser (Amoco), which coincides with the peak of the Cy5.5 absorbance. At 680 nm, the excitation contrast ratio for Cy5.5/Cy5 is 5.1. A suitable excitation filter is the Ealing 35-4068.

c. The emission contrast ratio between Cy5.5 and Cy5 peaks at 705 nm, approximately 3 nm to the red of the Cy5.5 intensity curve. The numerical value for S_(b) at this point is 4. If the Omega 700EFLP longpass emission filter is used, a contrast ratio of approximately 3 (averaged out to 800 nm) is expected. This, combined with the high excitation contrast, makes imaging Cy5.5 very clean.

d. At the expense of slightly lower emission contrast (this would not be significant) and some loss of intensity, a bandpass filter such as the Ealing 35-6345 could be substituted for the Omega 700EFLP. The potential advantage would be reduction of the infra-red background i.e., overall improved image contrast.

Imaging Cy7

Cy7 is the reddest dye of the set. The excitation and emission spectra are well separated from Cy5.5, and are well matched to the Omega 740DF25/770DRLPO2/780EFLP triplet. The Oriel 58895 is an appropriate IR blocker for Cy7.

Filters selected for imaging the DAPI, FITC, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 fluor set are summarized in the Table 2 below. None of these filter sets correspond to the filter sets supplied by manufacturers of conventional fluorescence microscopes as narrow band excitation and fluorescence detection is mandatory to achieve sufficient

TABLE 2 Epicube Filter Configuration (for 75 W Xe Arc Source) Excitation Emission Bandpass Dichroic Bandpass Fluor Filter Beamsplitter Filter IR Blocking DAPI Zeiss Zeiss Zeiss None 365 nm 395 nm >397 nm FITC Omega Omega Omega BG38 455DF70 505DRLP02 530DF30 Cy3 Omega Omega Ealing BG38 546DF10 560DRLP02 35-3722 Cy3.5 Ealing Omega Zeiss BG38 35-3763 590DRLP02 630/30 Cy5 Omega Omega Omega Oriel 58893 640DF20 645DRLP02 670DF32 Cy5.5 Ealing Omega Omega Oriel 58893 35-4068 DRLP02 700EFLP Cy7 Omega Omega Omega Oriel 58895 740DF25 770DRLP02 780EFLP

Characteristics of the microscope system are described in detail by Ballard S. G. et al. (J. Histochem. Cytochem. 41:1755-1759 (1993), herein incorporated by reference). A high pressure 75 W DC xenon arc (XBO) was used as an excitation source because of its approximately constant spectral power distribution. A Zeiss Axioskop microscope workstation equipped with a cooled CCD camera (Photometrics CH250) was employed. The objective lens was a 63×1.25NA Plan Neofluar which should be “plan” and apochromatic with a high numerical aperture. The filter sets selected were able to discriminate between the six fluors with a maximum contrast (Table 2). To minimize the crosstalk between fluorophores, the filter sets were selected on the basis of maximum spectral discrimination rather than maximum photon throughput. Image exposure times were varied to adjust for photon flux differences and flux excitation cross-sections. Narrow band excitation and fluorescence detection is mandatory to achieve sufficient contrast. Appropriate excitation and emission filter sets were used to optically discriminated these fluors (Table 2).

The combinatorial labeling strategy relies on accurate measurements of intensity values for each fluorophore. Critical features are accurate alignment of the different images, correction of chromatic aberrations, and specific quantitation of each Ifluorophore. Because simple manual image manipulation could not realize these demands new software was developed in our lab. This program comprises the following steps in sequential order. (1) correction of the geometric image shift; (2) calculation of a DAPI segmentation mask; (3) for each combinatorial fluor, calculation and subtraction of background intensity values, calculation of a threshold value and creation of a segmentation mask; (4) use of this segmentation mask of each fluor to establish a “Boolean” signature of each probe; (5) for each chromosome, display of the chromosome material next to the DAPI image; (6) create a composite gray value image, where each labeled object is encoded with a unique gray value; (7) final presentation of the results using a look-up-table (LUT) that assigns each gray value a particular color.

The above-described program was developed on the basis of an image analysis package (BDS-Image) implemented on a Macintosh Quadra 900. Image shifts caused by optical and mechanical imperfections were corrected by the alignment of the gravity center (center of mass) of a single chromosome in each image according to a procedure described by Waggoner, A. et aL (Methods Cell Biol 30:449-478 (1989)) and modified (du Manoir, S. et al., Cytometry 9:4-9 (1995); du Manoir, S. et al., Cytometry 9:21-49 (1995), all herein incorporated by reference). The DAPI image was used to define the morphological boundary of each chromosome. Accurate chromosome segmentation was achieved by pre-filtering the images through a top-hat filter (Smith, T. G., et al., J. Neurosci Methods 26:75-81 (1988); modified in du Manoir, S. et al., Cytometry 9:4-9 (1995); du Manoir, S. et al., Cytometry 9:21-49 (1995)). The mode of the gray level histogram of the top-hat filtered DAPI image was used as the threshold. For each fluor background was eliminated by subtracting the mean interchromosomal fluorescence intensity from the image. The mean of the chromosomal fluorescence intensities was used to calculate the threshold for the individual segmentation mask of each fluor. Individual DNA targets were assigned distinct gray values depending on the “Boolean” signature of each probe, i.e. combination of fluors used to label this DNA probe. In a final step a look-up table was used to assign each DNA target a pseudocolor depending on this gray value, for display.

Switching the filter sets in excitation and emission path as well as the dichroic mirror was done manually, but a computer-controlled electromechanical solution will allow automation of the procedure.

3. Optical Combs

In an alternative embodiment, selective excitation of multiple fluors and analysis of fluorescence spectral signatures can be carried out using dispersion optics rather than wavelength-selective transmission filters. Such optics may be used to create filters of any passband characteristic, including short-pass, long-pass, single bandpass and multiple bandpass functions. In this method, a dispersion element (prism or grating) is used in conjunction with a wavelength-selective spatial filter to create the desired spectral response. The combination is referred to herein as a “comb filter.” Using a comb filter, the spectral distribution of the exciting light may be tailored for optimum simultaneous excitation of multiple fluors. The inverse comb filter may also be used to selectively block from the CCD camera only the wavelengths used for excitation: the remaining wavelength intervals (corresponding to the gaps between teeth of the comb) are available for spectral analysis of the fluorescence emitted by the fluors. This analysis constitutes the spectral signature.

4. Interferometers

In lieu of using the optical filters described above, an interferometer may be used in conjunction with an epi-fluorescent microscope. A light source for excitation of fluorescence that is either coherent (e.g. an Argon laser) or incoherent (e.g. a Mercury arc lamp) may be used. A Mercury-Xenon mixed gas arc lamp is preferred due to its intense Mercury lines and broad Xenon visible and near-infrared continuum.

Although any of a variety of interferometer designs (such as Michelson interferometer) may be employed, the use of a Sagnac interferometer is preferred. The Sagnac interferometer has a larger acceptance angle, greater entendue, and is less sensitive to alignment, vibration, and temperature variations than a similar Michelson interferometer.

The Sagnac interferometer is a common path interferometer. An interferometer consists of two or more interfering beams of light. In a common path interferometer there are two beams each traveling the same path but in opposite directions. The optical paths are produced by reflecting light through beamsplitters, for example.

Multiple beam interferometers operate by dividing the optical energy from a light source into two substantially equal beams of light. The two beams of light are combined after one is permitted to pass through a sample and the interference pattern (the changes in intensity of the combined light caused by the interference of two beams) is detected.

In the Sagnac interferometer, the light source is also divided into two substantially equal parts. Changing the angle of incidence of light on the beamsplitter, (by rotation of the interferometer, or rotation of an optic, such as a galvanometer driven mirror within the interferometer) causes the optical path length to be changed along one optical axis of the interferometer. This produces a fringe pattern along one axis of the detector, for example a CCD detector. The other axis of the detector can sample gray scale. As the optical path length is scanned, by rotating the interferometer or a mirror, the fringe pattern produces an interferogram at each pixel. The Fourier Transform of this interferogram yields the spectrum of light falling on that pixel of the CCD. Thus, an advantage of the Sagnac interferometer is that it produces an optical path difference across an entire field of view, rather than at a single point.

Disturbances, such as a small shift of one of the optical elements, effect both beams in the same way, and hence have no effect on the measurement. This lmechanical stability also makes the interferometer relatively insensitive to temperature changes as well. Thus, another advantage of the common path interferometer is its intrinsic stability. Sagnac interferometers and their use are well known (see, for example, U.S. Pat. Nos. 3,924,952; 4,410,275; 4,529,312; 4,637,722; 4,671,658; 4,687,330; 4,836,676 and U.S. Pat. No. 5,108,183).

In one implementation of a Sagnac interferometer (J. Bruce Rafert et al., “Monolithic Fourier-Transform imaging spectrometer”, Applied Optics, November 1995), the acceptance angle of the interferometer is determined to be:

q=2 n tan⁻¹(w/8a)

where w/a=tan 30°, w is the aperture width of the interferometer, a is the length of each leg, and n is the index of refraction of the interferometer glass. The input beam to the interferometer need not be collimated.

The interference pattern or interferogram is most preferably detected with a CCD camera (such as a Princeton Instruments frame transfer CCD camera) capable of 512×512 pixels or larger. Since the interferogram in a Sagnac interferometer has an angular dependence, each pixel of the CCD detector measures a small interval of the interferogram. The fringe spacing of the interferogram is set such that a pixel on the CCD detector can adequately sample the interferogram. The Optical Path Difference (OPD) that a pixel can span, in order to properly sample the interferogram is given by the relation:

OPD_(pixel)=λ_(min)/4

where λ_(min) is the shortest wavelength in the spectrum to be measured by the interferometer. This OPD_(pixel) determines the theoretical limit of the resolving power of the interferometer.

As the OPD is being changed by the rotating mirror, the interferogram is being moved across the CCD detector, such that the maximum optical path difference is then given by the relation,

OPD_(max) =N(OPD_(pixel))

where N is the linear dimension of the CCD detector in pixels. Each angular displacement of the light incident on the interferometer beamsplitter may then correspond to one or several OPD_(pixel's). And, in the case of a CCD detector, one frame of CCD data is required to sample this angular displacement.

Finally then, each pixel comprises an interferogram which contains within it information about the spectrum of light falling on the pixel, the intensity of light falling on that pixel, and the x and y coordinates of the pixel. The spectrum of light may be recovered from the interferogram by the use of a computational Fourier Transform algorithm.

In practice, because of the limited dynamic range of CCD's, typically about 10,000:1, the light used to excite the fluorescence must be blocked from entering the interferometer. This excitation light is often 10⁸ to 10¹² more intense than the fluorescence that is emitted from the sample. Without blocking by using optical filtering, this excitation light would saturate the CCD. However, this filter need only be fabricated so as to block the excitation, all other wavelengths may be allowed to pass.

In one embodiment, ultra violet (UV) light is used to excite the fluorescent probes. The UV light may be easily blocked with a long pass interference filter allowing the visible and near-infrared colors to pass through to the interferometer. This embodiment has the advantage that UV will excite many of the fluorescent dyes currently in use. This embodiment also has the advantage that it will allow better than 90% transmittance of the visible fluorescence to the interferometer. The disadvantage of UV is that it photobleaches the dyes faster than visible light.

Both the input and the output lens of the interferometer are preferably very high efficiency camera lenses, and do not significantly effect the efficiency of imaging. The focus of the image within the interferometer is most preferably adjusted so as to be constant for the variable powers of the zoom eyepiece, and thus a microscope having the characteristic of infinite image distance (such as Olympus AX70 microscope) are preferred.

The above-described interferometer possesses certain advantages over optical filters. One key advantage is that all the light emitted by fluorescence is theoretically available for detection, whereas the transmittance of an interference filter is limited. Another advantage is that since the filters do not have to be changed, there is no image shift due to the non-parallelism of filters.

E. Uses of the Present Invention

The capacity of the FISH (Fluorescent In situ Hybridization) methods and reagents of the present invention to detect and analyze chromosomal abnormalities, such as translocations, inversions, duplications, etc. can be used for a large number of applications.

1. Applications Relating to the Cytogenetic Diagnosis of Genetic Disease

Among the primary applications of the present invention is the cytogenetic diagnosis of genetic disease, such as the pre- or post-natal diagnosis of disease, complex tumor karyotyping, the analysis of cryptic translocations (especailly through the use of telomere-specific probes). It provides a novel method for automated chromosome identification and analysis. A large number of diseases (prenatal disease, cancers (especially BRCA1 or BRCA2 associated breast cancer), leukemias, Down's Syndrome, etc.) are characterized by rearrangements and other chromosomal abnormalities that can be discerned using the methods of the invention.

Chromosome karyotyping by conventional cytogenetic banding methods is both time consuming, expensive and not easily automated. The detection of recurring genetic changes in solid tumor tissues by karyotyping are particularly problematic because of the difficulty in routinely preparing metaphase spreads of sufficient quality and quantity and the complex nature of many of the chromosomal changes, which make marker chromosome identification based solely on banding patterns extremely difficult. Indeed, attempts to automate karyotype analysis over the past twenty years (e.g., pattern matching, eigen analysis) have failed because robust computer algorithms could not be developed to reliably decipher complex banding patterns, particularly those of extensively rearranged chromosomes.

It has been proposed that the next generation of cytogenetic techniques would be far superior by using bands that are defined molecularly by hybridization of probes or probe set s each labeled with a different color (Nederlof, P. M. et al., Cytometry 11:126-131 (1990); Nederlof, P. M. et al., Cytometry 13:839-845 (1992); Lengauer, C. et al., Hum Mol Genet 2:505-512 (1993)). This would provide a high versatility and would constitute a quantum leap well comparable to the introduction of chromosome banding and high resolution analysis of chromosomes in prometaphase. Advantages of the FISH karyotype are the instant identification of the chromosomal origin of marker chromosomes, double-minutes and homology staining regions (“HSRs”). Even “poor quality” chromosome spreads can be evaluated. If desired, one could design a probe set for particular applications or for particular clinical applications, e.g. hematologic diseases, pre- or postnatal diagnosis. The development of specific probe sets that stain particular regions of chromosomes (e.g. telomeric regions) for the identification of cryptic translocations would overcome limitations of the whole chromosome painting probes. Similarly, such probes could be used to generate multicolor “barcodes” on individual chromosomes thereby facilitating the automated analysis of karyotype. Probes can also be designed that would be specific for a particular arm of a chromosome, thereby permitting a molecular characterization of translocation breakpoints, hot spots of recombination, etc. Other applications would include rapid evolutionary studies, provided that the protocols for multicolor FISH on human chromosomes can be adjusted, as expected, for applications on other species.

2. Applications Relating to the Detection of Infectious Agents

The methods of the invention may also be used to assess the presence or absence of infectious agents (treponerna pallidum, rickettsia, borrelia, hepatitis virus, HIV, influenza virus, herpes, Group B streptococcus, diarrhea-causing agents, pathogens causing acute meningitis, etc.) in tissue, or in blood or blood products. This can be accomplished by employing labeled probes specific for such agents. Moreover, by employing serotype-specific probes, the methods of the present invention permit the rapid serotyping of such agents, or the determination of whether any such agents carry drug resistance determinants. The methods of the present invention may be used to assess chromosomal abnormalities caused by exposure to radiation (such as personnel exposed to the radioactivity of nuclear power plants).

The methods of the present invention may be used to quantitate microorganisms that are difficult to propagate (such as anaerobic microorganisms involved in periodontal disease). The methods of the present invention provide a means for the rapid diagnosis of acute bacterial meningitis. Just as one can employ serotype-specific probes to perform serological analysis, one can employ probes that are specific to particular drug resistance determinants, and thereby rapidly determine not only the presence and identity of an infectious agent, but also its susceptibility or resistance to particular antibiotics.

3. Additional Applications

The methods of the present invention further permit simultaneous mapping of a large number of different DNA probes. With this technique the analysis of chromosomal number and architecture in individual intact cells becomes accessible. Interphase cytogenetics is already possible with small region specific probes, e.g. YAC-clones. The accuracy of such analysis could be increased by a three dimensional analysis using a laser scanning microscope, or more preferably, a CCD camera system with a Z-axis stepping motor coupled with 3-dimensional (3-D) image deconvolution software. Suitable 3-D image deconvolution software is obtainable from Imstar Corp. (Paris, France) or Scanalytics, Inc. (Boston). In addition, the use of such scanning microscope systems would ultimately allow one to visualize all whole-chromosome painting probes, and telomere- or centromere-specific probes sets, in interphase nuclei and questions relating to intranuclear chromosomal organization as a function of developmental status, cell cycle or disease state could be addressed. Different models about the chromosomal organization in interphase nuclei could finally be explored. Although conventional laser scanning microscopes currently do not allow the excitation of some of the fluorophores used, other, more appropriate fluors or devices may be employed including 3 dimensional deconvoluting imaging systems.

Extended to non-mitotic cells, the methods of the present invention enable one to examine chromosome architecture or quantitate the chromosome contents of nuclei in single hybridization experiments. Questions relating to intranuclear chromosomal organization as a function of developmental status, cell cycle or disease state can accordingly be addressed. In addition, the ability to quantitatively assess the levels of multiple mRNAs or proteins in a single cell or to determine if they exhibit different intracellular distributions could prove extremely useful in addressing a myriad of interesting biological questions. The multiparametric imaging of the present invention does not merely increases the throughput of information, it also makes more efficient use of the biological material. Thus, it can reveal spatial and temporal correlations as well as mosaicisms that might otherwise be difficult to establish reliably. Since the intracellular distribution of mRNAs and protein is not known a priori to be spatially distinct, as in the case for the intra-nuclear chromosome domains, it will not be possible to use the combinatorial labeling strategies in these experiments. However, many mRNA and protein antigens can be spectrally resolved and detected using a multiplex format with n fluors. Thus, for the first time the intracellular distribution of oncoproteins or tumor suppressor proteins can be determined within the same cell simultaneously.

F. Automated Karyotypic Analyses

One aspect of the present invention relates to automated, preferably computer-facilitated, karyotypic analyses. As described above, in one embodiment of the invention, the chromosomes of a particular karyotype are pseudo-colored to thereby facilitate the assignment of the chromosomes, or the recognition of translocations, deletions, etc. In one sub-embodiment thereof, the digitized images of the chromosomes may be stored in a computer-readable storage device (such as a magnetic or optical disk) to facilitate their comparison with other chromosomal images or their transmission and study. In this regard, probes may be employed that are translocation specific or specific to sub-chromosomal elements or regions, such that the pseudocolorized chromosomes. or chromosomal elements are displayed to the investigator as chromosomal images depicting a cytogenetic banding pattern (for example, the cytogenetic banding pattern of the metaphase chromosomes of the patient). The position and sizes of individual bands is preferably digitized and stored so that an image of the chromosome may be stored on a computer. Similarly, the precise position of any translocation or other karyotypic abnormality can be discerned and stored.

The methods of the present invention thus permit karyotypic analyses to be conducted more widely and more accurately than was previously feasible. The present invention may thus be used to systematically correlate karyotypic abnormalities with disease or conditions. For example, karyotypes of asymptomatic individuals can be obtained and evaluated in light of any subsequent illness (e.g., cancer, Alzheimer's disease, etc.) or condition (e.g., hypertension, atherosclerosis, etc.) in order to permit a correlation to be made between a patient's karyotype and his or her predisposition to different diseases and conditions. Similarly, karyotypes of individuals having diagnosed diseases or conditions can be obtained and evaluated in light of the extent of any subsequent progression or remission of the disease or condition so as to permit a correlation to be made between a particular karyotype and the future course of a disease or condition.

In a further sub-embodiment, a computer or other digital signal analyzer may be employed to orient and arrange the chromosomal images as well as assigning and identifying the chromosomes of the karyotype. Thus, a computer or other data processor will, upon assigning a particular chromosome to a particular designation (for example, upon assigning that a particular chromosomal image is the image of the chromosome 7 of the karyotype being evaluated), group the assigned chromosome with its homologue (e.g., the second chromosome 7 of the patient's karyotype) and generate, via a printer, monitor, or other output means, an ordered array of chromosomal images (such as one in which each autosomal chromosome is paired with its homologue, and in which the sex chromosomes X and Y are paired together).

In one sub-embodiment, the chromosomal images of such arrays will be the pseudocolorized images discussed above. Alternatively, such psudocoloring may be internal to the process of assigning chromosomal identity, and not displayed in the output of the computer or digital signal analyzer. Rather, in this sub-embodiment, the output generated will be the light-microscope visible cytogenetic banding pattern of the metaphase chromosomes of the patient whose karyotype is being evaluated. In a further sub-embodiment, a scale (in Morgans or other suitable units) will be superimposed upon the chromosomal images.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

Example 1 Combinatorial Labeling of Chromosoines

In order to test the feasibility to produce 24 colors chromosome painting probes representing the 22 autosomes and the two sex chromosomes were used. The DNA probes used were generated by microdissection.

Microdissected probes (National Center for Human Genome Research, Bethesda, Md.) give a very uniform labeling of the target region. The detailed protocols for microdissection and PCR amplification are described by Telenius et al. (Telenius, H. et al., Genes, Chromosomes & Cancer 4:257-263 (1992); Telenius, H. et al., Genomics 13:718-725 (1992); Meltzer, P. S. et al., Nature Genetics 1:24-28 (1992); Guan, X. Y. et al., Hum Mol Genet 2:1117-1121 (1993); Guan, X. Y. et al., Genomics 22:101-107 (1994); Guan, X. Y. et al., Hum Genetics 95:637-640 (1995), all herein incorporated by reference). For some chromosomes different DNA-probes for the p- and the q-arms were available, namely 2, 4, 5, 10, 11, 16, 18, and Y. For all other chromosomes microdissected probes painting the entire chromosome were used.

The first member of the set of preferred fluors, DAPI, was used as a genera] DNA counterstain. The remaining fluors: fluorescein, Cy3, Cy3.5 (emission and excitation spectra are between Cy3 and Cy5), Cy5 (Mujumdar, R. B. et al., Cytometry 10: 11-19 (1989), and Cy7 (emitting to the red of Cy5 (Ernst, L. A., et al., Cytometry 10:3-10 (1989)), were used to combinatorially label different probes (Table 3). Distinctive features of these dyes are high extinction coefficients, quantum yields, and photostabilities. Fluorescein is a xanthene dye with an extinction coefficient around 70,000 L/mol cm and quantum yields in optimal buffers around 0.7. The respective values for cyanines are 1-200,000 and 0.3 (Waggoner, A., Methods in Enzymology 246:362-373 (1995)).

After microdisection, the probes were subjected to a PCR amplification and labeled by nick translation. Fluorescein (Wiegant, J. et al., 19:3237-3241 (1991)), Cy3, and Cy5 were directly linked to DUTP for direct labeling. Cy3.5 and Cy7 were available as avidin or antidigoxin conjugates for secondary detection of biotinylated or digoxinigated probes. They were synthesized using conventional N-succinamide ester coupling chemistry. For each probe one to three separate nick translation reactions were necessary, each with a single labeled fluor-labeled triphosphate or biotin or digoxigenin (Table 3).

TABLE 3 Chromosome Fluor 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y FITC X X X X X X X X X X X Cy3 X X X X X X X X X X Cy3.5 X X X X X X X X X X X Cy5 X X X X X X X X X X Cy7 X X X X X X X X X X

As expected probes labeled with equal amounts of different fluors did not give equivalent signal intensities for each fluor reflecting the fact that the filter sets were selected to maximize spectral discrimination rather than photon throughput. In order to diminish signal intensity-differentials, probe concentrations for the hybridization mix had to be established careflully in a large number of control experiments. Hybridization conditions were optimized for these multiplex probes. Thus, probes were denatured and hybridized for two to three nights at 37° C. to metaphase chromosome spreads in a conventional 50% formamide hybridization cocktail. The slides were washed at 45° C. in 50% formamide/2 ×SSC three times followed by three washes at 60° C. in 0.1×SSC to remove excess probe. After a blocking step in 4×SSC/3% bovine serum albumin for 30 min at 37° C. the biotinylated probes were detected with avidin Cy3.5 and the dig-labeled probes with anti-dig-Cy7. Fluorescein-dUTP, Cy3-dUT'P, and Cy5-dUTP did not require any immunological detection step. After final washes at 45° C. with 4×SSC/0.1% Tween 20 three times, mounting medium and a coverslip were applied and the hybridization signals from each fluor imaged using the filters sets listed in Table 3.

FIG. 1 provides a schematic illustration of the CCD camera and microscope employed in accordance with the present methods.

FIG. 2 shows the raw data from a karyotypic analysis of chromosomes from a bone marrow patient (BM2486). Adjacent to each source image is a chromosome “mask” generated by the software program. In FIG. 2, panels A and B are the DAPI image and mask; panels C and D are FITC image and mask; panels E and F are Cy3 image and mask; panels G and H are Cy3.5 image and mask; panels I and J are Cy5 image and mask; and panels K and L are Cy7 image and mask.

FIGS. 3A and 3B show the identification of individual chromosomes by spectral signature. FIG. 3A is the same photograph as FIG. 2, except that it is gray scale pseudocolored. FIG. 3B displays the karyotypic array of the chromosomes. The exceptional power of the methods of the present invention are illustrated by the ease with which the translocation of chromosomes 5 and 8 are identified in FIGS. 3A and 3B, relative to conventional non-chromosome specific karyotype analysis.

The above experiment demonstrates that five fluors can be spectrally discriminated to produce at least twenty four different colors. The combinatorial labeling schemes need not be as complex as previously thought, because using 5 fluors for probe labeling, only 9 painting probes need to be labeled with as many as three fluors. A sixth fluor for probe labeling would allow up to 63 possible fluor combinations. Such a high number of different targets will not be required for most applications, but would allow the selection of combinations with the best spectral signature. These above-described protocols allow highly reliable and reproducible multicolor FISH.

Example 2 Rapid Analysis of Multiple Oral Bacteria in Mixed Cell Populations Using Multiparametric Fluorescence In situ Hybridization (M-FISH)

In situ hybridization has been recognized as having potential application as a means of analyzing individual bacterial cells in the absence of culture. DeLong, E. F. et al. (Science 243:1360-1363 (1989)), for example, used oligonucleotide probes complementary to the 16s ribosomal RNA (rRNA) sequences to differentiate an archaebacterium (Methanosarcina acetivorans) from the eubacteria Bacillus megaterium and Proteus vulgaris. Van Den Berg, F. M. et al. (J. Clin. Pathol. 42:995-1000 (1989)) used total genormic DNA to detect Campylobacter pylori in stomach tissue. Amann, R. et al. (Nature 351:161-164 (1991)) detected Holospora obtiusa in the macronucleus of the protozoan Paramecium caudatum.

Prior to the present invention, however, the use of in situ hybridization to differentiate microorganisms in clinical specimens with mixed populations, especially in a quantitative fashion, was difficult and time consuming. This is particularly true of bacteria with fastidious growth requirements, in which culture methods may result in the selective enrichment of a subset of the organisms present in the specimen.

A. Analysis of Non-Cross Hybridizing Microbial Species

To further illustrate the present invention, the M-FISH methods of the present invention are used used to speciate bacteria involved in the microbial etiology of periodontal disease.

Several methods have previously been used to establish the microbial etiology of periodontal disease. Well over 200 bacterial species can be identified in one sublingual plaque sample using labor-intensive cultural methods (Dzink, J. L. er al., J. Clin. Periodont. 12:648-659 (1985); Moore, W. E. C.; J. Periodont. Res. 22:335-341 (1987); Moore, W. E. C., Infect. Immunity 38:651-667 (1987)). This diversity has hampered the association of specific microbes in the etiology of the various forms and stages of inflammatory periodontal diseases. Direct assessment, both qualitatively and quantitatively, of plaque bacteria would obviate the need for culturing and its attendant difficulties. DNA probes (French, C. K. et al., Oral Microbiol. Immunol. 2:58-62 (1986)) have been widely used in a blot hybridization format as a direct method for bacterial assessment. However, this method does not allow for the direct visualization of the bacterial or provide information on the relative abundance of a particular bacterial within a complex mixture.

In order to more simply illustrate the capacity of the invention to speciate microbial strains, a test for in situ hybridization specificity is conducted using a mixture of bacteria that are morphologically distinct and non-cross hybridizing. Using such a mixture, one can immediately differentiate positive hybridization from that of non-specific binding by correlating the correct morphology with the correct probe.

Thus, Fusobacterium nucleatum (25586), Porphyromonas gingivalis (33277), Eikenella corrodens (23834), Prevotella intermedia (25611), and Actinobacillus actinomycetemcomitants (29522) were obtained from the American Type Culture Collection, Bethesda, Md., and Capnocytophaga species, C. ochracea (C25) and C. gingivalis (DR2001) were obtained from The National Institute of Dental Research, Bethesda, Md.

Total genomic DNAs were isolated from F. nucleatum, P. gingivalis, E. corrodens, P. intermedia, A. actinomycetemcomitans, C. gingivalis, and C. ochracea. using the procedure of Chassy, B. M. et al., Appl. Environ Microbiol. 39:153-158 (1980)) as modified by Donkersloot, J. A. et al., J. Bacteriol 162:1075-1078 (1985)). Each DNA was labeled with 32p and tested for crosshybridization with each of the other DNAs by dot blot hybridization. In all cases, the total genomnic DNA probes show specificity for the bacteria from which it was derived; no hybridization with any of the other bacterial DNAs was detected. Since the genomic DNA probes were highly specific in the dot blot assays, they were used directly for the in sinti identification of these bacteria in reconstructed mixtures.

Total genomic bacterial DNAs are labeled by nick translation using biotin-11-dUTP (BIO), digoxigenin-11-dUTP (DIG) or dinitrophenol-11-dUTP (DNP) as described by Lichter, P. et al., Hum. Genet. 80:224-234 (1988), herein incorporated by reference). Unincorporated nucleotides are removed using a Sephadex G-50 spin column equilibrated with 10 mM Tris-HCI/1 mM EDTA/0.1% SDS, pH 8.0. Labeled DNAs (2-6 μg) are ethanol precipitated and redissolved in 100% deionized formamide. F. nucleatum DNA is labeled with BIO-11-dUTP (BIO) and detected with Cascade Blue avidin; A. actinomycetemcomitans DNA is labeled with DNP-11-UTP (DNP) and detected indirectly with an FITC conjugated antibody; and E. corrodeyis DNA is labeled with DIG-11-dUTP (DIG) and detected with a Rhodamine labeled anti-DIG antibody.

To prepare slides for in situ hybridization, small aliquots (1-2 ml) of cultured bacteria are centrifuged at 1,200×g in an HBI microcentrifuge. The cell pellet is suspended in phosphate buffered saline (PBS) and adjusted to approximately 10³ bacterial per μl. Aliquots of both pure cultures and synthetic mixtures are covalently bound to activated glass slides prepared as described by Maples, J. A. (Amer. J. Clin. Pathol. 83:356-363 (1985), herein incorporated by reference). Alternatively, bacterial are spotted directly onto positively (+) charged slides and air dried. Suitable slides are commercially available from Fisher Scientific, Pittsburgh, Pa. (cat. #12-550-15), and retain all the bacterial strains listed above throughout the in situ hybridization procedure with high efficiency. All slides are then fixed in Carnoy's B Fixative (Ethanol: Chloroform: Acetic Acid, 6:3:1) for 5 minutes.

The bacteria are hybridized simultaneously with the three differentially labeled genomic DNAs. Each sample is hybridized using 15 μl of hybridization solution which contains 50 ng of labeled probe and DNAse-treated salmon sperm DNA (15 μg) in 50% (vol/vol) deionized formamide/2×SSC (0.3 M sodium chloride/0.03 M sodium citrate, pH 7.0)/5% dextran sulfate. The solution is applied to the sample, covered with a coverslip and sealed with rubber cement. Both bacterial DNA and labeled DNA probe are denatured by heating at 80° C. for 5-8 minutes in a oven. These steps are sufficient to permeabilize the bacteria for probe assessability.

The DNAs were allowed to reassociate by incubating the slides at 37° C. for 18 hrs in a moist chamber. Posthybridization washings, blocking and detection were as described by Lichter et al. (7). Briefly, the slides were washed 3× in 50% forinamide/2×SSC at 42° C. for 5 minutes, and then washed 3× in 0.1×SSC at 60° C. for 5 minutes. The slides were then incubated in a solution of 3% bovine serum albumnin/4×SSC (blocking solution) for 30 minutes at 37° C.

Biotinylated probes were detected using fluorescein isothiocyanate-(FITC)-avidin DCS (Vector Laboratories, Burlingame, Calif., 5 μg/ml) Cascade Blue-avidin (Molecular Probes Inc., Eugene, Oreg., 10 μg/ml)) or Rhodamine-avidin (Boehringer Mannheim, Indianapolis, Ind., 10 μg/ml).). Digoxigenin-labeled probes were detected using FITC or Rhodamine conjugated sheep anti-digoxigenin Fab fragments (Boehringer Mannheim, 2 μg/ml). Dinitrophenol labeled probe was detected by incubating with rat anti-DNP antibodies (Novagen, Madison, Wis., 1:500 dilution)) and then with goat anti-rat FITC-conjugated antibodies (Sigma, St. Louis, Mo., 1 μg/ml). In some experiments, bacterial DNA was counterstained with 4,6-diamidino-2-phenylindole (DAPI) at a concentration of 200 μg/ml.

For detection, the fluorochrome-conjugated antibodies or avidin are diluted into a solution of 4×SSC/1% BSA and 0.1% tween-20 (200 μl/slide) and incubated with the sample at 37° C. for 30 minutes in the dark. The slides are then washed 3 times in 4×SSC/0.1% tween-20 at 42° C. prior to viewing via epifluorescence microscopy.

Epifluorescence microscopy is conducted using a Zeiss Axioskop-20 wide-field microscope with a 63× NA 1.25 Plan Neofluar oil immersion objective and a 50 W high pressure mercury arc lamp. Images are projected with a Zeiss SFL-10 photo-eyepiece onto a cooled charged-coupled device (CCD) camera (Photometrics CH220; 512×512pixel array). Effective magnification is set by changing the microscope-camera distance, using a bellows. The 8-bit greyscale images are recorded sequentially using DAPI, FITC and Rhodamine filter sets (manufactured by C. Zeiss, Inc., Germany) to minimize image offsets. Camera control, greyscale image acquisition and image pre-processing are done using an Apple Macintosh 11x computer preferably running custom software (Dr. Marshall Long; Yale University). Images are pseudocolored, aligned and merged using a Macintoch 11ci computer equipped with an accelerated 24-bit graphics system (SuperMac Spectrum 24 PDQ). The merging software, “Gene Join Max Pix” (Yale University) is preferably employed. This software assigns a user definable pseudocolor and greyscale value to each of the source images, and then generates an output (“merged”) image based on the most intense source image at each pixel location. Thus, objects with higher scaled brightness (e.g., probe signals) override the DAPI counterstain signals in the corresponding image location. Objects displayed in merged image retain the color assigned to them and therefore appear distinct and easily identifiable. Merged images were photographed directly from the computer monitor.

A mixed sample of F. nucleatum, E. corrodens and A. actinomycetemcomitans, in a 1:1:1 ratio was used as a test sample. As can be seen in FIG. 4, the three bacteria are distinguishable on the basis of morphology when stained with DAPI.

FIG. 4 shows the differentiation of bacteria by in situ hybridization. Panels A-E represent in situ hybridization assay results on a laboratory derived mixture of three bacteria (F. nucleatum, A. actinomycetemcomitans and E. corrodens) using a mixture of genomic DNA probes for each organism. Panel A shows all the bacteria present in the microscope field detected by (DAPI), a general DNA binding fluorophore. F. nucleatum is seen using Cascade Blue detection (panel B), A. actinomycetemcomitans with FITC detection (panel C) and E. corrodens using Rhodamine detection (panel D). Panel E is a computer-merged composite of panels B-D showing the differentiation of each bacteria in the sample. Note that a single A. actinomyetemcomitans cell is detected in a clump of E. corrodens (panel E) that could not be identified by DAPI staining (panel A). Panel F is a similar analysis of a mixture of C. gingivalis (Rhodamine) and P. intermedia (FITC) when hybridized with a combination of genomic DNA probes for those organisms. Panel G shows an in situ hybridization assay for a combination of seven different bacteria hybridized with a mixture of seven genomic DNA probes. F. nucleatum (1), E. corrodens (2) and A. actinomycetemcomitans (3) are shown in blue (Cascade Blue detection). C. gingivalis (4) and C. ochracea (5) are shown in red (Rhodamine detection), whereas P. intermedia (6) and C. gingivalis (7) are seen in yellow (FITC detection). Phase contrast microscopy was used to assist in determining morphological differences between E. corrodens and A. actinomycetemcomitans which were not readily evident through the hybridization generated signals. Panel H represents an in situ hybridization assay for the presence of A. actinomycetemcomitans (FITC detection) in a plaque sample obtained from a patient with localized juvenile periodontist.

When assaying for Cascade Blue, only the F. nucleatum bacteria (FIG. 4, panel B) were seen, when assaying for FITC only A. actinomycetemcomitans bacteria (FIG. 4, panel C) were visible, and when assaying for Rhodamine, only the E. corrodens bacteria (FIG. 4, panel D) were positive. The three separate fluorophore images were then merged to generate the composite image that is shown in FIG. 4, panel E. Each of the morphologically distinct bacteria exhibit the correct fluorescence specificity.

The four additional bacterial species are included in subsequent experiments. These bacteria are combined pairwise such that each mixture again would differ morphologically. FIG. 4, panel F shows a combined analysis of P. intermedia (BIO label and FITC detection) and C. gingivalis (DIG label and Rhodamine detection). An analysis of C. ochracea and P. gingivalis, whose morphology is similar to the P. intertnedia and C. ochracea and P. gingivalis, whose morphology is similar to the P. intermedia and C. gingivalis pair, is also performed. In both cases, absolute specificity of the probe for its cognate bacteria is demonstrated.

Since the above experiments indicate that none of the bacteria cross-hybridized and that each species of bacteria, as grouped, could be distinguished by morphology as well as by hybridization via the binding of a specific fluorochrome, it was reasoned that all seven bacteria could be detected in a single mixed sample. The experimental design was to label the DNA from the bacteria within each of the three morphologically distinct groups with the same “reporter” (BIO, DIG or DNP). The three groups would be initially differentiated by hybridization and the specificity of the fluorescence signal. The bacteria within a group could then be differentiated on the basis of morphology.

To demonstrate this capacity of the present invention, P. interrnedia and C. gingivalis DNAs were labeled with DIG, F. nucleatum, A. actinomycetemcomitans, and E. corrodens DNAs were labeled with BIO whereas P. gingivalis and C. ochracea DNAs were labeled with DNP. All seven probes were then combined and used for in situ hybridization on mixed bacterial samples containing all organisms (FIG. 4, panel G). In FIG. 4, panel G, the F. nucleatum, E. corodens, A. actinomycetemcomitans group is shown using Cascade Blue (BIO detection) and its members are numbered 1, 2 and 3 respectively; the P. gingivalis and C. ochracea group is seen with Rhodamine (DIG detection) and these bacteria are numbered 4 and 5 respectively; the C. gingivalis, P. intermedia group is identified with FITC (DNP detection) and they are numbered 6 and 7 respectively. All seven bacteria are clearly differentiated using the combination of hybridization fluorescence and morphology. While morphological identification could be made simply on the basis of size, shape and color of the hybridization signal, more definitive morphological characterization is made by examination of the sample using phase-contrast optics.

To determine how few hybridization-positive bacteria could be detected in a sample, 4.0 μl of E. corrodens containing 10⁵ bacteria/μl is mixed with 1.0 μl of serial dilutions of A. actinomycetemcomitans and the 5 μl mixture applied to slides. The sample is then tested for the presence of A. actinomycetemcomitans using a BIO-labeled probe for that bacterium. Following the hybridization, the total bacteria on the slide are visualized using Propidium Iodide and the hybridization-positive bacteria are detected using Avidin-FITC. The results of this experiment show that single A. actinomycetemcomitans cells could be detected in the presence of an excess of at least 10⁴ E. corrodens cells. Thus, it is apparent that hybridization positive bacteria can be identified readily even when they constitute only a minor fraction of a mixed cell population.

Clinical samples are not usually as free of contaminating debris as laboratory derived mixtures of bacteria. To test the potential utility of M-FISH for the analysis of clinical specimens, a dental plaque sample from a patient with localized juvenile periodontitis, in which prior studies (Slots, J., Dent. Res. 84:1 (1976); Slots, J. et al., J. Clin Periodontal. 13:570-577 (1986); Zambon, J. et al., J. Periodontal. 54:707 (1983)) had suggested the involvement of A. actinomycetemcomitans as the microbial pathogen is employed. FIG. 4, panel H shows the results of an in situ hybridization experiment with this patient's sample using BIO-labeled A. actinomycetemcomitans DNA and detecting with FITC-conjugated avidin. As can be seen, a large number of A. actinomycetemcomitans or other closely related organisms were detected. Quantitative studies indicate that more than 50% of the individually identifiable bacteria seen in the specimen hybridized with the A. actinomycetemcomitans DNA probe. Cultural studies on this sample showed the presence of A. actinomycetemcomitans.

All of the above experiments employ a standard overnight hybridization time (˜18 hours) to differentiate the bacteria of interest. Even though such analyses can often be done faster than the traditional methods of culturing and biochemical testing, a number of experimental parameters could be adjusted to give a more rapid assay. It is well known that hybridization time is directly related to probe concentration. Thus, by increasing probe concentration, hybridization time can be proportionately reduced. A mixture of F. nucleatum and A. actinomycetemcomitans is therefore analyzed using a 10-fold higher concentration of probe than previously used (from 50 ng to 0.5 μg per slide). At the higher probe concentration no specific or non-specific hybridization is observed with undenatured probe and bacterial DNA. However, when the probe and bacterial DNA are denatured simultaneously, and the washing cycles begun immediately, some of the DNAs hybridized during the time necessary for subsequent sample processing.

In this experiment, in which 0.5 μg of A. actinomycetemcomitans probe is introduced to a sample containing both A. actinomycetemcomitans and F. nucleatum bacteria using a 2.5 minute incubation at 37° C. prior to initiating the washing cycles, the hybridization signal is as strong and as specific as that observable in experiments utilizing 50 ng of probe and hybridizing for 18 hours. Furthermore, by reducing the blocking and antibody detection times to 20 minutes each, a total assay time of 1.5 hours is achieved. Further reduction in assay times clearly can be obtained using probe DNA directly labeled with the fluorophore detector.

The above experiments provide a relatively rapid and simple method for identifying individual bacteria in mixed microbial populations using M-FISH. The method provides a quantitative assessment of specific microorganisms without the necessity of culturing. The technique can be applied to samples containing only a limited number of organisms and a single hybridization-positive bacteria can be detected quite easily in a 10⁴⁻ fold excess of hybridization-negative bacteria. In addition, as exemplified by the overlapping and “hidden” A. actinomycetemcomitans cell among the group of E. corrodens cells (FIG. 4, panel E), bacterial specified that are not clearly identified by a non-specific analysis (i.e., DAPI staining of DNA, FIG. 4, panel A) can be detected by the hybridization-specific assay. Since both the hybridization specificity and the morphological characteristics of bacteria can be assessed simultaneously by this procedure, the number of different bacterial species can be increased still further by judicious selection of probe labeling and fluorophore detector combinations.

However, morphological characteristics are not always a reliable indicator of bacterial identity since many different bacterial species appear morphologically similar and some are pleomorphic depending on the culturing methods. For example, A. actinomycetemcomitans are generally coccobacillary in clinical specimens but can assume rod and even filamentous forms upon in vitro passage and in clinical specimens. Thus, additional fluorescence colors would preferably be employed to expand the number of different bacteria that can be analyzed simultaneously and identified unequivocally.

The intensity of the hybridization signal is extremely strong using either whole genomic DNA or subtracted (suppressed) probes. Although data is collected by digital imaging, hybridization signals can be detected readily by eye and recorded by conventional photographic methods. However, the sensitivity and photon counting capabilities of CCD-camera based imaging system offer considerable advantages for further refining microbial analysis by in situ hybridization. Detection of single copy sequences for specific toxin or drug resistance genes in individual bacteria should be feasible; individual human and murine genes have been visualized, both in metaphase chromosomes and interphase nuclei by FISH (Lichter, P. et al., Hum. Genet. 80:224-234 (1988)). The level of cross-homology between bacterial strains also can be rapidly assessed by quantitating the photon output of the hybridization signal from individual bacteria in a specimen. Finally, and most importantly, digital imaging is essential to fully exploit the potential of combinatorial fluorescence.

As exemplified here, total genomic DNA can often provide adequately specific probes, thus eliminating the usual cloning and screening efforts necessary for probe production. Although not required in these studies because of the absence of cross hybridization between the bacterial genomes, suppression hybridization techniques (Lichter, P. et al., Hum. Genet. 80:224-234 (1988)) can be used to squelch cross-hybridization between organisms that share partial sequence homology. For example, C. orchracea and C. sputigena exhibit 22-23% sequence homology (Zambon, J. et al., J. Periodontal. 54:707 (1983)) yet specific hybridization to C. orchracea can be obtained with genoric DNA by preannealing the C. ochracea probe with an excess of C. sputigena DNA just prior to the in situ hybridization reaction.

In sum, total genomic DNAs from seven anaerobic and facultative oral bacteria recognized as pathogens in periodontal disease (Fusobacterium nucleatum, Porphyromonas (Bacteroides) gingivalis, Prevotella intermnedia, Eikenella corrodens, Actinobacillus actinomycetemcomitans, Capnocytophaga gingivalis and Capnocytophaga ochracea) are labeled non-isotopically and hybridized in situ to reconstructed bacterial mixtures. Each probe is hybridized uniquely to the bacterium from which it was derived. Three bacterial strains are differentiated simultaneously by labeling DNA with different reporter groups and visualizing the hybridization signal with reporter-specific detector proteins labeled with different fluorophores. By assessing bacterial morphology in conjunction with the hybridization signal each bacteria in a mixture of all seven organisms can be identified. The total assay time is as short as 1.5 hours. A dental plaque sample from a patient with localized juvenile periodontitis, an oral mixed microbial infection, reveals numerous hybridization positive bacteria when probed with DNA from A. actinomycetemcomitans, the putative microbial pathogen for this disorder. This example demonstrates the usefulness of the in situ hybridization methods of the present invention for the identification of individual non-cross hybridizing bacteria in mixed cell populations.

The seven bacterial species analyzed above are facultative or obligate anaerobes that are often found in association with inflammatory periodontal disease. The role of these microorganisms is uncertain due to the lack of rapid and reliable methods to identify specifies. Direct analysis of oral microbial specimens circumvents biases imposed by culture methods and may allow a better understanding of the role of these bacteria in the pathogenisis of periodontal disease. Data on the detection of A. actinomycetemcomitans in a specimen from a patient with localized juvenile periodontitis indicates that the above-described in situ hybridization procedure facilitates such studies. It also complements traditional culture methods for the identification of a broad spectrum of microorganisms, especially notoriously slow growing bacterial such as anaerobes or mycobacterium. For example, swabs taken from early culture plates may be streaked on a slide and assayed by in situ hybridization using probes for suspected pathogens of interest.

The present invention permits one to increase further the number of simultaneously detectable bacteria without increasing the number of available fluorophores by using combinatorial fluorescence imaging (Nederlof, P. M. et al., Cytometry 11:126-131 (1990)). By labeling a single probe with two (or more) different reporter molecules, the resultant hybridized probe will be detected by more than one of the fluorescent detectors and the signal will appear in more than one of the separate flurorescence images. When these separate pseudocolored images are merged into a composite image (as in FIG. 4 panels E, F and G), fluorescence signals appearing at the same site on two images will be “blended” and generate a new pseudocolor that is distinguishable from either of the originals. In this fashion, three fluorophores have been used combinationally to visualize seven probes simultaneously, each appearing as a distinct color (Ried, T. et al., Proc Natl Acad Sci (USA) 89:1388-1392 (1992)). By applying this combinatorial fluorescence imaging strategy to bacterial identification, one can obtain a distinct pseudocolor for each of the seven bacterial species analyzed above.

The above example demonstrates the feasibility of differentiating multiple bacterial species (such as those found in the periodontal microflora) using multiparametric fluorescence inl situ hybridization (M-FISH) and digital imaging microscopy. By monitoring both hybridization signal specificity and bacterial morphology, seven different bacteria are readily and simultaneously distinguished in a mixed population using only three distinct fluorophores. Such methods are readily applicable to the detection of bacteria (as well as viruses, and/or lower eukaryotes) that may be present in other clinical samples (in cluding those that contain complex mixtures of microflora (e.g., stool, saliva, throat swabs, sputum, vaginal secretions, etc.) and those that are normally (e.g., central spinal fluid (meningitis), blood (sepsis), etc.) or non-clinical samples (food products, water reservoirs, ecosytems, etc.). Furthermore, by expanding the number of spectrally resolvable fluors used in combinatorial fashion, the number of different types of bacteria, viruses and/or lower eukaryotes that can be analyzed in a mixed population can be markedly increased.

B. Analysis of Cross Hybridizing Microbial Species

As indicated above, using differentially labeled total genomic DNA probes, the in situ hybridization and digital imaging fluorescence microscopy methods of the present invention can simultaneously differentiate seven microorganisms in a laboratory derived mixed sample. This technique provides both single cell detection sensitivity and the ability to correlate hybridization signal specificity with microbial morphology. The relative abundance of a specific organism in a complex mixture or bacteria, can be readily assessed and a single hybridization positive bacteria can be detected in an excess of 10⁴ other bacteria. Moreover, the assays can be completed in less than 1.5 hours. However, none of the genomic DNAs from the seven bacteria used in the above study exhibited cross-hybridization.

To illustrate the capacity of the present invention to differentiate among related (i.e., cross-hybridizing) microflora, a study is performed using Capnocytophaga DF 1 strains: C. gingivalis (DR2001), C. sputigena (D4), and C. ochracea (C25) Such strains exhibit cross-hybridization (Rubin, S. J., Eur. J. Clin. Microbiol. 3:253-257 (1984)). The strains are obtained from the NIH Institute of Dental Research, Bethesda, Md. Bacterial DNA is isolated as described above. Probes are made as follows: total genomic bacterial DNAs is labeled by nick translation using biotin-11-dUTP (BIO), digoxigenin-11-dUTP (DIG) or dinitrophenol-11-dUTP (DNP) as described by Lichter, P. et al. (Hum. Genet. 80:224-234 (1988)). Unincorporated nucleotides are removed using a Sephadex G-50 spin column equilibrated with 10 mM Tris-HCl/1 mM EDTA/0.1% SDS, pH 8.0. Labeled DNAs (2-6 μg) are ethanol precipitated and redesolved in 100% deionized formrnamide.

The genus Capnocytophaga encompasses a group of fusiform, gram-negative, capnophilic, fermatative, gliding bacteria (Ledbetter, E. R. et al., Arch. Microbiol. 122:17-27 (1979)) that are common inhabitants of the oropharyngeal flora (Holdeman, L. V. et al., J. Periodon. Res. 20:475-483 (1985)). C. gingivalis, C ochracea and C. sputigena have been implicated in gingivitis in children (Moore, W. E. C. et al., Infect. Immun. 46:1-6 (1984)) and periodontitis in juvenile diabetics and adults (Dzink, J. L et al., J. Clin. Periodont. 12:648-659 (1985); Mashimo, P. A. et al., J. Periodont. 54:420-430 (1983); Slots, J. et al., J. Dent. Res. 63:414-421 (1984)). These three bacteria are members of the first of three groups of “Dysgonic Fermenters” (DF-1) defined by the Center for Disease Control (Speck, H. et al., Zbl Bakt. Hyg. A. 266:390-402 (1987)). The DF-2 group contain species designated as C. canimorsus and C. cynodeami, although these bacteria appear both phenotypically and genetically distinct from the Capnocytophaga species of the DF-1 group (Brenner, D. J. et al., J. Clin. Microbiol. 27:231-235 (1989)). Few of the microbes comprising the DF-3 group have been characterized, however it is thought that they are closely related to the Capnocytophaga DF-1 species (Gill, V. J. et al., J. Clin. Microbiol. 29:1589-1592 (1991)). Members from all three DF groups have been identified as the etiologic agents found in some cases of septicemia, joint infections and endocarditis (Arlet, G. et al., Ann. Biol. Clin. 44: 373-379. (1986); Juhl, G. et al., J. Infect. Dis. 149:654 (1984); Matlow, A. et al., J. Infect. Dis. 152:223-234 (1985); Mosher, C. B. et al., J. Clin. Microb. 24:161-162 (1986); Parenti, D. M. et al., J. Infect. Dis. 151:140-147 (1985); Ratner, H., Clin Microbiol 6:10 (1984); Rummens, J. L. et al., J. Clin. Microbiol. 75:376 (1985); von Graevenitz, A., Eur. J. Clin. Microbiol. 3:223-224 (1984)). The increased spectrum of oral and intra-oral infections caused by Capnocytophaga DF-1 species shows the pathogenic potential of this genus and may reflect differences in clinical significance of the respective species in medically important infections and periodontal disease (von Graevenitz, A., Eur. J. Clin. Microbiol. 3:223-224 (1984)). Conventional methods of identifying bacteria from oral sources requires first, isolation of single bacterial colonies from the oral microbial milieu, and second, the assignment of isolates to genus and species based upon cellular morphology, gram stain, fermentation of carbohydrates and hydrolysis of various substrates. These procedures require considerable time and expertise, taking upwards of 2 weeks to identify single species from clinical samples. Such conventional methods often fail to isolate Capnocytophaga from oral samples because these bacteria are slow growing, fastidious as to growth conditions and are therefore overgrown by other oral flora (Mashimo, P. A. et al., J. Periodont. 54:420430 (1983); Rummens, J. L. et al., J. Clin. Microbiol. 75:376 (1985)). Moreover, such methods may fail to discriminate C ochracea and C. sputigena unambiguously due to phenotypic similarities (Gill, V. J. etal., J. Clin. Microbiol. 29:1589-1592 (1991)).

Recent methods of microbial identification using enzymatic tests (Heltsberg, O. et al., Eur. J. Clin. Microbiol. 3:236-240 (1984); Kristiansen, J. E. et al., Euir. J. Clin. Microbiol. 3:236-240 (1984)), specific immunologic or molecular probes (Murray, P. A. et al., Oral Microbiol Immunol. 6:3440 (1991); Smith, G. L. F. et al., Oral Microbiol Immunol. 4:41-46 (1989)) have been used with some success for identifying bacteria in clinical oral samples. With the exception of enzymatic techniques, these methods have not been applied to the identification of Capnocytophaga spp. Enzymatic tests still require the isolation of pure colonies of bacteria, whereas immunologic and molecular methods can be performed directly on an oral sample, thereby overcoming the difficulties of isolating pure colonies of fastidious microorganisms. Generally, molecular methods rely on the use of DNA probes, formatted as a dot-blot hybridization assay, for the identification of a particular microorganism in a clinical sample (Parenti, D. M. et al., J. Infect. Dis. 151:140-147 (1985)). A major drawback of this method is that DNA from 10³ to 10⁶ microorganisms of interest are required for such assays, therefore bacteria in low abundance in a mixed population are likely not to be detected. In addition, cross-hybridization, which is common in closely related species, can impede identification to the species level.

It had been previously reported (Williams, B. L. et al., Arch. Microbiol. 122:35-39 (1979)) that C. gingivalis, C. ochracea, and C., sputigena DNAs exhibit up to 22% sequence homology, based on reassociation kinetics. Using conventional in situ hybridization methods, such extensive homology would appear to preclude the use of total genome DNA probes for the speciation of Capnocytophaga strains and necessitate the production and characterization of species-specific subgenomic clones. However, one of the attributes of the computerized digital imaging camera system of the present invention is the ability to collect hybridization data in the form of quantitative fluorescence images and to process such image data with appropriate computer software.

The intensity of the hybridization signal (i.e., fluorescence photon output) from a bacteria (“bacteria A”) A will be greatest with a genornic DNA derived from that bacteria. Related strains which share some DNA sequences in common with a DNA probe of genomic DNA for bacteria A (genomic DNA probe A) will give less intense hybridization signals, which directly reflect the extent of sequence homology. Unrelated bacteria that share little or no sequence homology to bacteria A will yield virtually no fluorescence signal at all. Once the relative extent of DNA sequence homology between stains has been established, cross-hybridization noise can be electronically suppressed (preferably via appropriate computer software and image thresholding techniques) thus directly leading to bacterial identification.

Computer thresholding of fluorescence images is used to establish the extent of cross-hybridization (i.e., sequence homology) between the three Cagnocytophaga species. In this experiment, pure samples of each bacteria are separately hybridized with biotinylated DNA probes from all three species. For example, three slides are prepared that contain only C. ochracea cells. The first slide is hybridized in situ using BIO-labeled C. ochracea genomic DNA, the second slide is hybridized in situ using BIO-labeled C. gingivalis genomic DNA and the third slide is hybridized in situ using BIO-labeled C. sputigena genomic DNA. The slides are then washed and hybridization positive bacteria are identified using FITC-avidin.

C. ochracea and C. gingivalis did not crosshybridize with one another, however C. sputigena cross-hybridized with both C. gingivalis and C. ochracea. A computer analysis of the intensity of the fluorescent hybridization signals indicates that C. sputigena had significant sequence homology with C. ochracea (˜22%) but much less (˜5%) with C. gingivalis.

To demonstrate how image thresholding can be used for bacterial speciation, a slide with a mixture of C. sputigena and C. orchracea is hybridized with Bio-labeled genomic DNA from C. sputigena. To prepare such slides for in situ hybridization, small aliquots (1-2 ml) of cultured bacteria are centrifuged at 1,200×g in a HBI microcentrifuge. The cell pellet is suspended in phosphate buffered saline (PBS) and adjusted to approximately 10³ bacteria per μl. Aliquots of both pure cultures and synthetic mixtures are spotted directly onto commercially available (Fisher Scientific, cat #12-550-15) positively (+) charged slides and air dried. All slides are then fixed in Carnoy's B Fixative (Ethanol: Chloroform: Acetic Acid, 6:3:1) for 5 minutes.

Hybridization is accomplished using 15 μl of hybridization solution which contained 50 ng of labeled probe and DNAse treated salmon sperm DNA (15 μg) in 50% (vol/vol) deionized for-mamide/2×SSC (0.3 M sodium chloride/0.03 M sodium citrate, pH 7.0)/5% dextran sulfate. The solution is applied to the sample, covered with a coverslip and sealed with rubber cement. Both bacterial DNA and labeled DNA probe are denatured by heating at 80° C. for 5-8 minutes in an oven. These steps are sufficient to permeabilize the bacteria for probe assessability.

The DNAs are allowed to reassociate by incubating the slides at 37° C. for 2.5 min. in a moist chamber. Posthybridization washings, blocking and detection are conducted as described by Lichter, P. et al. (Hum. Genet. 80:224-234 (1988)). Briefly, the slides are washed 3 times in 50% formamide/2×SSC at 42° C. for 5 minutes and then washed 3 times in 0.1×SSC at 60° C. for 5 minutes. The slides are then incubated in a solution of 3% bovine serum albumin/4×SSC (blocking solution) for 30 minutes at 37° C.

Biotinylated probes are detected using fluorescein isothiocyanate-(FITC)-avidin DCS (Vector Laboratories, 5 μg/ml), Cascade Blue-avidin (Molecular Probes Inc., 10 μg/ml) or Rhodanrine-avidin (Boehringer Mannheim, 10 μg/ml). Digoxigenin-labeled probes are detected using FITC or Rhodamine conjugated sheep anti-digoxigenin Fab fragments (Boehringer Mannheim, 2 μg/ml). Dinitrophenol labeled probe is detected by incubating with rat anti-DNP antibodies (Novagen, 1:500 dilution)) and then with goat anti-rat FITC-conjugated antibody (Sigma, 1 μg/ml). In some experiments, bacterial DNA was counterstained with 4,6-diamidino-2-phenylindole (DAPI) at a concentration of 200 ng/ml. For detection, fluorochrome-conjugated antibodies or avidin are diluted into a solution of 4×SSC/1% BSA and 0. 1% tween-20 (200 μl/slide) and incubated with the sample at 37° C. for 30 minutes in the dark. The slides are then washed 3 times in 4×SSC/0.1% tween-20 at 42° C. prior to viewing.

Hybridization is visualized via epifluorescence microscopy using a Zeiss Axioskop-20 wide-field microscope with a 63×NA 1.25 Plan Neofluar oil immersion objective and a 50 W high pressure mercury arc lamp. Images are projected with a Zeiss SFL-10 photo-eyepiece onto a cooled charged-coupled device (CCD) camera (Photometries CH220; 512×512 pixel array). Effective magnification is set by changing the microscope-camera distance, using a bellows. The 8-bit greyscale images are recorded sequentially using DAPI, FITC and Rhodamine filter sets, manufactured by C. Zeiss, Inc., Germany to minimize image offsets. Camera control, greyscale image acquisition and image pre-processing are done as described above.

All bacteria are found to be hybridization positive in this experiment, although some appear to fluorescence more intensely than others. After the fluorescence image had been thresholded (using the Gene Join Max Pix software; Yale University) to remove components of the image that exhibited less than 25% of the maximum fluorescence intensity, only a subset of the original bacteria appear hybridization positive. These bacteria are all C. sputigena because only they exhibit 100% homology to the probe. This general strategy can be used to speciate multiple related microorganisms, provided the level of their sequence relatedness is known so that appropriate thresholding parameters can be applied. Bacteria exhibiting up to 40% sequence homology can be discriminated with genomic DNA probes using this technique.

The Gene Join Max Pix software can be used in a second way to achieve bacterial speciation with genomic DNA probes. This embodiment exploits the fact that when separate fluorescence images (e.g., fluorescein and rhodamine) are merged to form a composite image; the source image with the greatest fluorescent intensity (i.e., photon output) at each pixel location will be color-dominant in the merged image. This attribute permits the use of multiple differentially labeled genomic DNA probes simultaneously to probe a single or mixed bacterial population. Since each bacteria in a mixture will exhibit the greatest fluorescence signal with the genomic probe that is 100% homologous, the image merging process results in effective speciation.

This is demonstrated by the following experiment. A mixture of the three Capnocytophaga strains is hybridized using a 1:1:1 of all three genomic DNA probes, each labeled with a different reporter. C. ochracea DNA was labeled with DNP-11-dUTP for detection with FITC (yellow green), C. gingivalis DNA was labeled with biotin-11-dUTP for detection with Cascade Blue (blue) and C. sputigena DNA was labeled with DIG-11-dUTP for detection with Rhodamine (red). The image generated by Cascade Blue shows intense signals for some bacteria present in the sample, presumably C. gingivalis cells and weak signals for other bacteria. The FITC fluorescence image produced by the C. ochracea probe and the Rhodamine fluorescence imge produced by the C. sputigena probe also show both intense signals for some bacteria and weak signals for others, reflecting interspecies hybridization. However, each bacterium shows an intense signal in one of the three images and a weaker (or absent) signal in the other images (reflecting interspecies hybridization). Accordingly, through the use of thresholding (i.e., using the signal generated to determine which probe gave the more dominant signal with which bacteria), the method enables one to determine the species of each bacterium.

Thus, in contrast to traditional methods of culturing and testing biochemical products for the differentiation and/or speciation of a bacteria, that are often difficult and time consuming, the in situ hybridization methods of the present invention provide a rapid and simple method for the speciation of cross-hybridizing microorganisms, such as the oral Capnocytophaga. The basis of this method relies on the finding that all microorganisms within different genera and species have unique DNA sequences within their genome. Closely related organisms will have some DNA sequences in common, and the amount of such cross-hybridizing sequences will directly affect their ability to be differentiated by in situ hybridization.

In this analysis, the fluorescence images obtained from in situ hybridization with each detector (e.g., Rhodamine, FITC or Cascade Blue), which reflect the amount of hybridization of a bacterium to each of the genomic DNA probes, are digitalized via computer. If a bacteria has no cross-hybridizing sequence with any of the other bacteria it will hybridize only to its homologous DNA and thereby be detected by only a single detector and its presence will be found in only the image corresponding to that detector. However, if a bacteria does have cross-hybridizing sequences with one of the other bacteria, then it will be detected by the DNA probes from each bacteria and its presence will be seen in the corresponding images for each probe (detectors). However, the intensity of the signal generated in each image will be directly proportional to the amount of hybridization of a bacterium with a DNA probe. The corresponding images from each detector are pseudocolored and overlaid (merged) to form a composite image. Since any given pixel on the monitor can only be a single color, only the most intense signal at each pixel is shown, and displayed as the corresponding pseudocolor.

Whereas two of the Capnocytophaga species (C gingivalis and C ochracea) of the above experiment show no cross-hybridization, the third species (C sputigena) cross-hybridized with both of the preceding organisms. The amount of cross-hybridization as estimated by a computer analysis of the intensity of the hybridization signal following in situ hybridization is in good agreement with that found in the literature ( 22-23% for C sputigena and C. ochracea and 9-11% for C. sputigena and C. gingivalis (Heltberg, O. et al., Eur. J. Clin. Microbiol. 3:236-240 (1984)). The 22-23% crosshybridization of C. sputigena and C. ochracea is evident in the composite image within some of the C. sputigena cells, but not evident in the C. ohcracea cells. This may reflect differences in how the cross-hybridizing sequences are arranged within those genomes.

This example further demonstrates that for an in situ hybridization analysis, the DNA probe need not be totally specific. Cross-hybridizing sequences must only be reduced below the level necessary for showing specificity. The level of unique sequences necessary for specificity would desirably be ascertained for each bacteria of interest, since it would probably be affected by not only the total number of cross-hybridizing sequences, but also how those sequences are arranged within a genome. However, some microorganisms, such as Neisseria meningitis and N. gonorrhoea show greater than 80% sequence homology (Kingsbury, D. T., J. Bacteriol. 94:870-874 (1967)). Such large amounts of cross-hybridizing sequences may result in an ambiguous analysis using in situ hybridization with total genomic DNA probes that can be addressed by the methods of the present invention.

Thus, in sum, multiparametric fluorescence in situ hybridization (M-FISH) and computer assisted digital imaging microscopy have been used to develop a rapid method for differentiating C. gingivalis, C. ohcracea and C. Sputigena with total genomic DNA probes. Although these bacteria exhibit up to 22% sequence homology, cross-hybridization signals can be completely suppressed. Speciation of these three Capnocytophaga species can be achieved in as little as 30-90 minutes, markedly faster than the 2-3 weeks required using conventional microbiological methods. The general discrimination strategy reported here is applicable to the broad spectrum of microbial pathogens for which an extent of sequence homology is known.

Example 3 Synergistic Use of Nucleic Acid Amplification Technology in Concert with M-FISH Analysis

Nucleic acid amplification technology has greatly increased the ability to investigate detailed questions about the genotype or the transcriptional phenotype in small biological samples, and has provided the impetus for many significant advances in biology, especially in the field of genetics.

One aspect of the present invention concerns the use of the above-described multiparametric fluorescence in situ hybridization (M-FISH) methods in concert with one or more methods of nucleic acid amplification to facilitate the detection and characterization of mutations, chromosomal elements, chromosomal rearrangements, and infectious agents (e.g., viruses, proviruses, etc.) that may be present in small amount or proportion in a sample undergoing analysis. For example, through the use of fluorophore combinations designed for multiparametric color coding, a 6-fluor tagging approach permits the simultaneous identification of 63 different types of signals by virtue of their unique spectral signatures.

As described above, the M-FISH method of the present invention can distinguish and identify all of the chromosomes of a human karyotype. However, by amplifying a specific gene, allele or chromosomal element, and employing one or more labeled probes that are capable of specifically hybridizing to the amplified sequences, the invention permits the determination of whether a particular karyotype contains the gene, allele or chromosomal element in question. By way of illustration, the use of a probe specific for a deletion, rearrangement, insertion or other mutation characteristic of a genetic disease (e.g., hemophilia, cystic fibrosis, breast or other cancer, etc.) it is possible to diagnose whether a patient exhibits the genetic disease (e.g., to diagnose cystic fibrosis, etc.). Similarly, the invention permits one to determine whether a patient's chromosomes carry a recessive allele associated with genetic disease. Likewise, the invention permits one to determine whether a patient is predisposed to a disease by virtue of the presence of a genetic lesion (e.g., a mutation in the apoE, p53, rb, or brcal/2 genes) associated with a future disease state (such as Alzheimer's Disease, heart disease, cancer, etc.).

Similarly, as applied to the investigation of microbes (e.g., bacteria, viruses, and lower eukaryotes), the M-FISH method of the present invention can distinguish and identify the species of microorganisms present in a clinical or non-clinical sample. However, by amplifying a specific gene, allele or chromosomal element, and employing one or more labeled probes that are capable of specifically hybridizing to the amplified sequences, the invention permits the determination of whether a particular microorganism contains the specific gene, allele or chromosomal element. Thus, for example, by using a probe specific for an antibiotic resistance determinant, a cellular antigen, a toxin, etc., the combined use of M-FISH and nucleic acid amplification permits a determination of whether a particular microbial or viral strain is resistant to an antibiotic, or is pathogenic, or expresses a toxin. Such combination of methodologies thus permits the serotyping and subspeciation of pathogens without any requirement of culturing and/or purification.

The M-FISH methods of the present invention particularly concern probe sets and methods that are sufficient to characterize both the genotypic and phenotypic charateristics of micobes present in a preparation. A genotypic probe is one capable of specifically hybridizing to phylogenetically related species of microbes; hybridization of such a probe to nucleic acid of a preparation thus reveals whether a preselected microbe, or its cross-hybridizing (i.e., phylogenetically) related microbes are present in such a preparation. A phenotypic probe is one capable of specifically hybridizing to genes, or genetic elements associated with a particular phenotype (e.g., antibiotic resistance, toxin production, antigen presentation, etc.).

The invention particularly contemplates the multiparametric use of multiple fluors, and particularly concerns the embodiments in which 2, 3, 4, 5, 6 or more fluorophores are employed so as to permit the detection and/or characterization of 3, 7, 15, 31, 63 or more combinations of genotypic or phenotypic elements. Thus, for example, if five fluors are employed, 31 genotypic/phenotypic elements can be probed. Any combination of such elements can be employed. For example, 25 of such elements can be genotypic elements (thus permitting the identification of 25 different species of microbes), whereas 6 of such elements can be genotypic (for example permitting the determination of whether any of the 25 identified species are resistant to any of 4 antibiotics or present any of 2 toxins). Similarly, 10 of such elements can be genotypic elements (thus permitting the identification of 10 different species of microbes), whereas 21 of such elements can be genotypic (for example permitting the determination of whether any of the 10 identified species are resistant to any of 9 antibiotics, present any of 3 toxins, present any of 4 surface antigens, and express any of 5 genes).

Any of a wide group of bacteria (e.g., E. coli strains and strains of other enterics (e.g., Salmonella), Clostridria, Vibrio, Corynebacteria, Listeria, Bacilli (especially B. anthracis), Staphylococcus Streptococci (especially beta-hemolytic Streptococci and S. pneumoniae), Borrelia, Mycobacterium (especially M. tuberculosi); Neisseria (especially N. gonorrhoeae), Trepanoma, bacteria implicated in periodontal disease (e.g., Fusobacteria, Porphyromonas, Eikenella, Prevotella, Actinobacillus, and Capnocytophaga species), etc.), viruses (e.g., parvoviruses, papoviruses, herpesviruses, togaviruses, retroviruses (especially HIV), rhabdoviruses, influenza viruses, etc.), and lower eukaryotes (fungi (e.g., Dermatophytes; Pneumocystis, Trypanosoma; etc.), yeast, helminths, nematodes, etc.) can be detected and characterized using such a method.

Significantly, the combined use of a nucleic acid amplification method and the multiparametric fluorescence in situ hybridization methods of the present invention can be used to explore the quiescence or expression state of cells. Thus, by employing probes specific to RNA produced by, for example, growing cells, cells expressing tumor antigens or hormones, etc., the methods of the present invention can determine not merely the presence of tumor cells, but the extent of their malignancy. Such mRNA profiling may be conducted even in circumstances in which standard cDNA hybridization approaches may not be sensitive enough to detect changes in the concentration of low abundancy gene products.

While the combined use of a nucleic acid amplification method and the multiparametric fluorescence in situ hybridization methods of the present invention can employ any suitable amplification method or methods (e.g., Polymerase Chain Reaction (Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H. et al., EP 50,424; EP 84,796, EP 258,017, EP 237,362; Mullis, K., EP 201,184; Mullis K. et al., U.S. Pat. No. 4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; Saiki, R. et al., U.S. Pat. No. 4,683,194 and Higuchi, R. “PCR Technology,” Ehrlich, H. (ed.), Stockton Press, NY, 1989, pp 61-68), Strand Displacement Amplification Walker, G. T. et al., Proc. Nat!. Acad. Sci. (U.S.A.) 89:392-396 (1992); Walker et al., U.S. Pat. No. 5,270,184; Walker, U.S. Pat. No. 5,455,166); Ligase Chain Reaction (Segev, WO90/01069; Birkenmeyer, WO93/00447), etc.), the preferred method of amplification is the Rolling Circle Amplification (RCA) method (Caplan, M. et al., PCT Patent Application Publication No. WO 97/19193; herein incorporated by reference in its entirety).

Rolling Circle Amplification (RCA) is an amplification strategy in which nucleic acid amplification is driven by a DNA polymerase that can replicate circularized oligonucleotide probes with either linear or geometric kinetics, under isothermal conditions. In detail, RCA involves incubating at least one one rolling circle replication primer (RCRP) with at least one amplification target circle (ATC). The ATC comprises a single stranded circular DNA molecule that contains a region complementary to the RCRP, such that the RCRP can hybridize to the ATC and mediate amplification in the presence of a DNA polymerase.

In the presence of two primers, one hybridizing to the “+” strand, the other to the “−” strand of DNA, a complex pattern of DNA strand displacement ensues that generates 10⁹ or more copies of each circle in 90 minutes, enabling detection of point mutations in human genomic DNA. This expanding cascade of strand displacement and fragment-generation events is termed “DNA Hyperbranching,” and the special rolling circle amplification driven by two primers is termed “Hyperbranched-RCA” or “HRCA.” Using a single primer, RCA generates hundreds of tandernly linked copies of a covalently closed circle in a few minutes.

It is preferred to conduct HRCA using exo(−) Vent DNA polymerase, or the large fragment of Bst DNA polymerase (Aliotta, J. M. et al., Genet. Anal. 12:185-195 (1996); Thomas, D. C. et al., Clin. Chem. 43:2219 Abs 38 (1997); both herein incorporated by reference), and by the Sequenase 2.0 variant of T7 DNA polymerase. It had been shown that Sequenase supports rolling circle amplification of circular oligonucleotides, albeit relatively slowly (Fire, A. et al., Proc. Natl. Acad. Sci. (USA) 92:4641-4645 (1995); Liu, D. etal., J. Amer. Chem. Soc. 118:1587-1594 (1996)). The efficiency of RCA and HRCA reactions is increased by the addition of proteins that bind single-stranded DNA. The addition of E. coli single-strand binding protein (SSB) stimulates Sequenase-catalyzed HRCA, while phage T4 gene-32 protein stimulates Vent exo(−) catalyzed HRCA. In contrast, the Bst polymerase does not require such ssingle-strand binding proteins for maximal activity.

If matrix-associated, the DNA product remains bound at the site of synthesis, where it may be tagged, condensed, and imaged as a point light source. Linear oligonucleotide probes bound covalently on glass surfaces can generate RCA signals, whose color indicates the allele status of the target, depending on the outcome of specific, target-directed ligation events. Single molecule counting by RCA provides a powerful mutation detection method for studies using oligonucleotide arrays, and is particularly amenable for the analysis of rare somatic mutations.

In a further embodiment, the combined M-FISH/nucleic acid amplification methods of the invention (especially RCAIHRCA) may be used to detect circularizable oligonucleotides, called “padlock probes” (Landegren, U. et al., Methods 9:84-90 (1996); Landegren, U. et al., Ann Med. 29:585-590 (1997); (Nilsson, M. et al., Science 265:2085-2088 (1994); Nilsson, M. et al., Nat. Genet. 16: 252-255 (1997), all herein incorporated by reference) bound to single copy genes in cytological preparations.

Multiple applications exist for the above-described gap-fill reaction, in which target-complementary sequences are incorporated into circles by copying and covalent closure. In principle, any DNA sequence thus captured into a circular DNA may be amplified by RCA or HRCA. The reaction may thus be advantageously used in situations where it is desirable to interrogate the sequence incorporated into a padlock probe at some point after RCA. Longer sequences, such as mnicrosatellite repeats, should also be capable of being copied into circularizable probes for amplification and analysis. Subsequent to such copying, since there is little likelihood that rolling circle amplification will modify the number of repeats incorporated into a circularized probe (Fire, A. et al., Proc. Natl. Acad Sci. (USA) 92:46414645 (1995)), direct measurement of repeat size would be feasible.

In particular, any of three alternative HRCA/RCA strategies for allele discrimination utilizing ligation of circularizable DNA probes and rolling circle replication —“gap-probe RCA,” “polymerase-mediated gap-fill RCA,” and “ligase-mediated extension of immobilized probe RCA” —may be employed in concert with the M-FISH methods of the present invention. The polymerase mediated “gap-fill” reaction is preferred over the “gap probe” ligation reaction for solution studies of complex genomes. This is because the gap oligonucleotides, which cannot be washed away in a solution assay, are preferably used at relatively high concentrations for the ligation step, and may therefore interfere with the HRCA reaction, inducing the formation of amplicon artefacts. In contrast, the gap ligation reaction is considered preferable for allele analysis of DNA in cytological specimens. Here, the specificity of ligation should be enhanced relative to probes without a gap because three different sequence recognition events and two independent ligation events must occur before “padlock” closure. The third assay method, ligase-mediated extension of an oligonucleotide linked to a solid surface, provides a totally novel approach to quantify individual hybridization/ligation events and to score rare somatic mutations.

In a highly preferred embodiment of HRCA/RCA, the DNA generated by RCA is labeled with combinatorially labeled fluorescent DNP-oligonucleotide tags that hybridize at multiple sites in the tandem DNA sequence. The “decorated” DNA, labeled by specific encoding tags, is then condensed into a small object by cross-linking with a multivalent antibody (e.g., anti-DNP IgM). The wild-type specific primer generates RCA products which can hybridize to fluorescein-labeled DNP-oligonucleotide tags, while the mutant RCA products hybridize to Cy3-labeled DNP-oligonucleotides. The process of Condensation of Amplified Circles after Hybridization of Encoding Tags is termed “CACHET.” The use of CACHET, in concert with the M-FISH methods of the present invention is particularly preferred.

Example 4 Use of M-FISH Analysis to Evaluate Telomere Integrity

Conventional analyses of chromosomes using, for example, Giemsa banding of metaphase chromosomes have several salient deficiencies. In particular, karyotypic changes involving chromosome fragments that have very similar banding patterns can be impossible to resolve using this method (Saccone, E. et al., Proc. Natl. Acad. Sci. (USA) 89:4913-4917 (1992)). Such a finding is particularly true of the telomeric bands which are virtually all Giemsa negative and which are also the most gene-rich bands within the genome. The correct identification of telomeric regions is very important because telomeres may be involved in breakage and healing events that can result in terminal deletion, gene amplification or cryptic translocations.

Indeed, there is an increasing number of clinical cases that have been found to be the result of cryptic translocations such as hemoglobin H (Lamb, J et al., Lancet 2:819-824 (1989)), Cri-du-Chat (Overhauser, J. et al., Amer. J. Hum. Genet. 45:296-303 (1989)), Wolf-Hirschhorn (Altherr, M. R. et al, Amer. J. Hum. Genet. 49:1235-12342 (1991)), and Miller-Dieker lissencephaly syndrome (Kuwano, A. et al., Amer. J. Hum. Genet. 49:707-714 (1991)). In addition, cryptic translocations occur in up to 6% of the population with mild to moderate mental retardation who have no detectable chromosomal changes upon karyotyping. Many of these genetic changes go undetected using standard cytogenetic analysis.

As indicated above, the present invention provides an ability to simultaneously identify the twenty-four different human chromosomes in a metaphase spread by hybridizing a complete set of chromosome-specific DNA probes, each labeled with a different combination of dyes. One aspect of the present invention is the recognition that, through the use of telomere-specific probes, the methods of the present invention may be used to identify translocations that would be non-identifiable (i.e., cryptic) through the use of conventional methods. As such, the present invention provides, for the first time, a simple screening test to assess the integrity of telomeric regions using a single hybridization reaction. Such multiplex hybridization assays significantly improve the ability to detect terminal deletions and cryptic chromosomal rearrangements, and thereby facilitate the identification of structural abnormalities that elude detection with conventional cytogenetic banding or multicolor whole-chromosome painting methods. Thus, this aspect of the present invention extends prior efforts in both karyotyping technology (see, e.g., Speicher, M. R. et al., Nature Genet 12:368-375 (1996); Speicher, M. R. et al., Bioimaging 4:52-64 (1996); Schrock, E. et al., Science 273:494-497 (1996)) and telomere integrity assay technology (see, e.g., Ledbetter, D. H., Amer. J. Hum. Genet. 51:451-456 (1992)); all herein incorporated by reference).

To illustrate this capacity of the invention, a set of YAC clones containing human DNA sequences specific for the sub-telomeric region of each chromosome arm is assembled. This probe set employed a microdissected probe for the short arm of the Y chromosome (Guan, X. Y., Nature Genet. 12:10-11 (1996), herein incorporated by reference) because no suitable sub-telomeric YAC for the short arm could be identified. Similarly, because of extensive cross-hybridization, the probe set did not include probes for the p arms of the acrocentric chromosomes 13, 14, 15, 21 and 22. The probes are combinatorially labeled such that the telomeric regions of these chromosomes are visualized in different pseudocolors based upon their unique fluorophore composition.

The p arm and q arm telomere proximal probes for each chromosome are labeled with the same “color code,” for example both chromosome 1 telomeres are labeled in color “a,” while the chromosome 2 telomeres are labeled in color “b,” etc. Thus, any cytogenetically cryptic chromosome translocation occurring on a non-acrocentric chromosome are visualized in the metaphase spreads as a chromosome harboring 2 colors rather than a single color per chromosome. Although the 24 color telomere integrity assays utilize a complex probe mixture, such probe cocktails can be hybridized with high reproducibility. Such hybridization is visualized, as described above, with, for example, an epifluorescence microscope equipped with an appropriate filter set and a cooled CCD-camera, etc. The fluors and high contrast filters are the same as those described above. In brief, fluorescein (FLU), Cy3, and Cy5 are linked to dUTP for direct labelling; Cy3.5 and Cy7 are available as avidin or antidigoxigenin conjugates for secondary detection of biotin- or digoxigenin-labelled probes. For each probe, 1-3 separate nick translations are performed.

The telomeric probe set may be composed of a mixture of YACs, Half-Yacs (Vocero-Akbani, A. et al., Genomics 36:492-506 (1996); Macini, R. A. et al., Genomic Res. 5:225-232 (1995)), and CEPH-YACs (Chumakov, I. M. et al., Nature 377:175-297 (1995)), chosen to contain the most telomeric genetic markers. Half-YACs are generated from specialized YAC libraries enriched for telomeric sequences. The term “half-YACs” refers to the fact that one of the vector telomere sequences of the molecule is provided by human DNA rather than yeast DNA. Half-YACs contain sub-telomere repeats that are known to reside on different chromosomes thus, in some instances, they hybridize to multiple telomeres. However, specific M-FISH signals can be obtained from half-YACs, for example through the use of DNA amplification (using, for example, PCR with Alu primers to reduce the relative representation of repeat sequences); addition of Cot-1 DNA to the hybridization cocktail will suppress hybridization of any residual repetitive sequences. Suitable YACS, half-YACs and/or CePH-YACs are described by Vocero-Akhani, A. et al. (Genomics 36:492-506 (1996)), by the National Intitutes of Health and Institute of Molecular Medicine Collaboration (Nature Genet 14:86-89 (1996)) and by Bray-Ward, P. et al. (Genomics 36:1-14 (1996)); all of which references are herein incorporated by reference. The fluors used to label telomeric regions are shown in Table 4. In Table 4, the references are (1) Vocero-Akbani, A. et al., Genomics 36:492-506 (1996); (2) National Intitutes of Health and Institute of Molecular Medicine Collaboration (Nature Genet 14:86-89 (1996)); (3) Bray-Ward, P. et al. (Genomics 36:1-14 (1996)); (4) Bray-Ward, P. et al. (Genomics 36:104-111 (1996)); (5)Guan, X. Y., Nature Genet. 12:10-11 (1996)), all herein incorporated by reference.

TABLE 4 Evidence for Most Distal Telomeric Insert STS on Localization/ Size Integrated Flpter Telomere FISH Probe Label Library Source (kb) Mapl (min-max) Reference  1p TYAC179 (HTY3222) Flu/Cy3 HDK-TYACL 200 sJCW15 Half-YAC 1  1q yRM2123 Flu/Cy3 HR-TYACL 270 Half-YAC 2  2p TYAC47 (HTY3047) CY5/Cy7 HDK-TYACL 350 sAVA22 Half-YAC 1 695h7 CEPH-L 1350 D2S359 0.01-0.06 3 955c11 CEPH-L 1280 D2S304 0.01-0.06 3  2q yRM2112 CY5/Cy7 HR-TYACL 240 Half-YAC 2 TYAC75 (HTY3118) HDK-TYACL 75 sSMMI Half-YAC 1 933c7 CEPH-L 1690 D2S159 0.93-0.95 3  3p 852b3 Flu/Cy7 CEPH-L 1100 D3S1270 0.00-0.05 3  3q TYAC162 (HTY3205) Flu/Cy7 HDK-TYACL 250 Sava54 Half-YAC 1  4p 764h1 Cy3.5/Cy5/Cy7 CEPH-L 400 3104 3  4q 946a3 Cy3.5/Cy5/Cy7 CEPH-L 1120 d4s2930 0.97-1.00 3  5p 789d2 Cy3/Cy7 CEPH-L 1630 D5S676 3  5q TYAC139 (HTY3182) Cy3/Cy7 HDK-TYACL 75 Half-YAC 1 887f10 CEPH-L 945 D5S498 0.91-1.00 3  6p 870d6 Flu/Cy3/Cy7 CEPH-L 1260 D6S344 0.00 4  6q 820f9 Flu/Cy3/Cy7 CEPH-L 1050 D6S281 0.92-0.95 3  7p 626g11 Cy3.5/Cy7 CEPH-L 290 D7S531 3  7q yRM2000 Cy3.5/Cy7 HR-TYACL 240 Half-YAC 2 TYAC109 (HTY3152) HDK-TYACL Half-YAC 1  8p yRM2205 Cy3/Cy5 HR-TYACL 250 Half-YAC 2 931b2 CEPH-L D8S264 0.00-0.04 3  8q yRM2053 Cy3/Cy5 HR-TYACL 170 Half-YAC 2 TYAC48 (HTY3048) HDK-TYACL 200 2058VI Half-YAC 1  9p 822e7 Flu/Cy3/Cy5 CEPH-L 1640 D9S178 3  9q 908a4 Flu/Cy3/Cy5 CEPH-L 900 D9S1872 0.86-0.91 3 10p TYAC95 (HTY3138) Flu/Cy5 HDK-TYACL Half-YAC 1 10q TYAC93 (HTY3136) Flu/Cy5 HDK-TYACL 250 Half-YAC 1 11p yRM2209 Cy3.5/Cy5 HR-TYACL 125 D11S2071 Half-YAC 2 11q 790g9 Cy3.5/Cy5 CEPH-L 1060 D11S968 0.95-1.00 3 12p 922c8 Cy3/Cy3.5 CEPH-L 1390 D12S1455 0.01-0.05 3 12q TYAC175 (HTY3218) Cy3/Cy3.5 HDK-TYACL 250 Half-YAC 1 13q yRM2067 Cy3/Cy3.5/Cy5 HR-TYACL Half-YAC 2 14q yRM2006 Cy3.5 HR-TYACL 200 Half-YAC 2 15q 877h4 Flu/Cy3.5/Cy7 CEPH-L 610 WI-5214 3 16p 927a8 Cy7 CEPH-L 120 D16S423 0.02-0.06 3 16q TYAC135 (HTY3178) Cy7 HDK-TYACL 100 Sava47 Half-YAC 1 17p 898a10 Flu CEPH-L 570 wi-5436 3 17q 965f1 Flu CEPH-L 1470 CHCLGATA4 3 FOR 18p yRM2102 Flu/Cy3/Cy3.5 HR-TYACL 220 Half-YAC 2 18q yRM2050 Flu/Cy3/Cy3.5 HR-TYACL 290 Half-YAC 2 19p 872g3 Cy3 CEPH-L 590 WI-9127 3 19q 926g5 Cy3 CEPH-L 1440 D19S887 3 20p 761d12 Flu/Cy3 CEPH-L 1330 AFMA049Y 3 D1 20q 793a9 Flu/Cy3 CEPH-L 1620 D20S173 3 21q yRM2208 Flu/Cy3.5/Cy5 HR-TYACL 290 Half-YAC 2 22q TYAC104 (HTY31476) Cy3/Cy3.5/Cy7 HDK-TYACL 75 Sava39 Half-YAC 1 Xp 827e10 Cy5 CEPH-L 920 wi-3553 3 Xq 883h10 Cy5 CEPH-L 1240 wi-7878 3 Yq MDP Flu/Cy5/Cy7 NIH 5

In order to test the reproducibility of specific signals with half YAC probes, each is carefully tested in single color experiments on normal metaphase spreads from at least five different donors. These studies permit the assessment of polymorphisms as well as the frequency or cross-hybridizations to other sub-telomeric repeats. Suitable half-YACs perform very reproducibly; in a few cases where occasional cross-hybridization occur, the nonspecific signals usually had very low fluorescence intensities and thus can be easily discriminated from a real signal.

CEPH-YACs were selected to contain the most telomeric genetic markers. Because the distance from the true telomeric end of the chromosome is unknown, these probes were tested by FISH for their distal band location. For six telomeric regions (2p, 2q, 5q, 7q, 8p, 8q) the hybridization of a single sub-telomeric YAC yielded low fluorescence intensities. Therefore two (5q, 7q, 8p, 8q) or three (2p, 2q) subtelomeric YACs were pooled in order to increase the signal. Alu-fingerprinting analysis was used to confirm that the YACs overlap.

A modified version of the previously published M-FISH software for whole chromosome painting probes was used for probe discrimination (Speicher, M. R. et al., Bioimaging 4:52-64 (1996); Schrock, E. et al., Science 273:494-497 (1996)); Yale University). In brief, to correct image shifts caused by optical and mechanical imperfections, whole chromosome painting probes, one for each fluor used in the experiment, are hybridized simultaneously with the pool of sub-telomeric YACs. Pixel shift correction of the chromosome painting probes is done as described above, however the chromosome paints are not shown on the final images to facilitate the color discrimination of the sub-telomeric probes. Instead of calculating a specific threshold for each fluor, based on the whole chromosome paint signals, the terminal regions of the chromosomes are specifically analyzed. Chromosome ends are identified and segmentation masks with different thresholds are calculated and checked for regions with increased fluorescence intensities. This is necessary because the simultaneous hybridization of multiple small region-specific probes results in signals that are often in different focal planes. In the absence of Z-axis optical sectioning and image merging, this results in some YAC probes giving significantly reduced fluorescence intensity values. If a specific telomeric region is not labeled with a particular fluor, no increased fluorescence intensity peaks is observed in at least one of the segmentation masks. The calculated fluor segmentation masks were not overlayed with the DAPI segmentation mask that delineates the chromosomal boundaries (since some of the telomeric FISH signals can lie outside the DAPI segment mask). Individual YAC-clones first are assigned distinct gray values depending on the boolean signature of each probe or the combination of fluors used to label it (Speicher, M. R. et al., Bioimaging 4:52-64 (1996); Schrock, E. et al., Science 273:494497 (1996)). A look-up-table then is used to assign each DNA target a psuedocolor depending on this gray value. Finally, this pseudocolored image was overlayed onto the DAPI-stained chromosome image that was assigned a light blue color.

In order to optimize the experimental parameters for hybridizing such a large number of combinatorially labeled YAC clones simultaneously, the multiplex telomere probe set is hybridized to normal metaphase spreads from peripheral blood lymphocytes. Similar to the results described above obtained with whole chromosome painting probes, YAC probes that are labeled with equal amounts of different fluors do not always give equal signal intensities for each fluor. To diminish signal intensity differentials, probe concentrations for the hybridization mix and a reliable combinatorial labeling scheme are therefore established by control experiments. YACs yielding large fluorescence signals are preferentially labeled with three different fluors while YACs yielding rather weak signals were labeled with one fluor only.

Using such probes, a 24 color telomere integrity assay is conducted on karyotypes of 10 normal male and female donors. No indication of polymorphisms are detected thereby suggesting that cryptic translocations are extremely infrequent in normal populations. A typical metaphase spread is observed (FIG. 5A), and a normal karyotype based on the boolean signature of our subtelomere probes is attained as expected (FIG. 5B).

In the process of testing the 24 color telomere integrity assay, the probe set is hybridized on metaphase spreads from a patient with a myeloproliferative disorder. Karyotyping using G-bands reveals trisomy 8 as the only detectable cytogenetic change. This is verified by M-FISH using chromosome specific painting probes. The telomere integrity assay, however, yields an intriguing hybridization pattern on the chromosomes 8. The trisomy 8 is verified in all 10 metaphase spreads analyzed. However, on 8 of these 10 metaphase spreads a split telomere signal is observed on two chromosomes 8, indicating an inversion in this region (FIG. 6). This analysis demonstrates the power of this new technique to detect structural abnormalities that are undetectable by standard cytogenetic methods or 24-color techniques using chromosome specific painting probes.

Cases where the application of the telomere-integrity assay may be very rewarding include evaluating karyotypes of patients with mental retardation, a combination of mental retardation and dysmorphic features, and cancer cells that are known to have a high genomic instability. The latter cells should be highly apredisposed to sub-telomeric translocation events. Screening for cryptic translocations can not be done efficiently with chromosome specific painting probes because they were not designed to detect subtle deletion or rearrangment events.

In a further embodiment of the above methods, the telomere-specific probe sets can be improved using PCR-assisted chromatography (Craig, J. et al., Hum. Genet 100:472-476 (1997), herein incorporated by reference). This tool removes repetitive DNA, including the polymorphic repetitive sequences from half-YACs or other M-FISH probes, and thereby facilitates the generation of probes that will hybridize specifically in the absence of Cot-I suppressor DNA.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims. 

What is claimed is:
 1. A method of achieving simultaneous visualization of at least 24 different genetic targets by multiparametric fluorescence in situ hybridization comprising: (A) contacting a preparation of chromosomes that has been previously treated to render it in single-stranded form with a set of combinatorially and distinguishably labeled probes, wherein said set of combinatorially and distinguishably labeled probes are combinatorially linked or coupled to 5 fluorophores; (B) hybridizing said set of combinatorially and distinguishably labeled probes with said preparation of chromosomes; and (C) simultaneously visualizing said at least 24 different genetic targets.
 2. The method of claim 1, wherein said set of combinatorially and distinguishably labeled probes are oligonucleotide probes.
 3. The method of claim 1, wherein said set of combinatorially and distinguishably labeled probes are microdissected probes.
 4. The method of claim 1, wherein said set of combinatorially and distinguishably labeled probes are probes for a sub-region of a target chromosome.
 5. The method of claim 1, wherein said set of combinatorially and distinguishably labeled oligonucleotide probes is capable of detecting a telomere.
 6. The method of claim 1, wherein said set of combinatorially and distinguishably labeled probes is capable of detecting a centromere.
 7. The method of claim 1, wherein said set of combinatorially and distinguishably labeled probes is DNA probes.
 8. The method of claim 1, wherein at least one member of the set is combinatorially labeled with a fluorophore selected from the group consisting of the fluorophores DAPI, FITC, Cy3, Cy3.5, Cy5, and Cy7.
 9. The method of claim 8, wherein at least two members of the set are combinatorially labeled with a fluorophore selected from the group consisting of the fluorophores DAPI, FITC, Cy3, Cy3.5, Cy5, and Cy7.
 10. The method of claim 9, wherein at least three members of the set are combinatorially labeled with a fluorophore selected from the group consisting of the fluorophores DAPI, FITC, Cy3, Cy3.5, Cy5, and Cy7.
 11. The method of claim 10, wherein at least four members of the set are combinatorially labeled with a fluorophore selected from the group consisting of the fluorophores DAPI, FITC, Cy3, Cy3.5, Cy5, and Cy7.
 12. The method of claim 8, wherein the fluorophore is conjugated to a ligand capable of binding to a modified nucleotide residue.
 13. The method of claim 12, wherein said ligand is selected from the group consisting of avidin, streptavidin, biotin-binding antibodies and digoxigenin-binding antibodies. 